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Abstract:

The present disclosure provides isolated monoclonal antibodies that
specifically bind to CXCR4 with high affinity, particularly human
monoclonal antibodies. Nucleic acid molecules encoding the antibodies of
this disclosure, expression vectors, host cells and methods for
expressing the antibodies of this disclosure are also provided.
Immunoconjugates, bispecific molecules and pharmaceutical compositions
comprising the antibodies of this disclosure are also provided. This
disclosure also provides methods for detecting CXCR4, as well as methods
for treating various cancers, inflammatory disorders and HIV infection
using an anti-CXCR4 antibody of this disclosure.

Claims:

1. A monoclonal antibody, or an antigen binding portion thereof, which
cross-competes for binding to human CXCR4 with a reference antibody or
reference antigen-binding portion thereof, wherein the reference antibody
or portion thereof comprises: (a) a heavy chain variable region
comprising amino acids having the sequence set forth in SEQ ID NO: 25 and
a light chain variable region comprising amino acids having the sequence
set forth in SEQ ID NO: 29; (b) a heavy chain variable region comprising
amino acids having the sequence set forth in SEQ ID NO: 26 and a light
chain variable region comprising amino acids having the sequence set
forth in SEQ ID NO: 30; (c) a heavy chain variable region comprising
amino acids having the sequence set forth in SEQ ID NO: 27 and a light
chain variable region comprising amino acids having the sequence set
forth in SEQ ID NO: 31; or (d) a heavy chain variable region comprising
amino acids having the sequence set forth in SEQ ID NO: 28 and a light
chain variable region comprising amino acids having the sequence set
forth in SEQ ID NO: 32; and further wherein the monoclonal antibody or
portion thereof comprises a heavy chain variable region comprising amino
acids having a sequence derived from a human VH 3-48 germline
sequence as set forth in SEQ ID NO:49 and a light chain variable region
comprising amino acids having a sequence derived from a human VK L15
germline sequence as set forth in SEQ ID NO:50.

2. A method of modulating CXCR4 activity in a cell comprising contacting
the cell with the antibody, or antigen-binding portion thereof, of claim
1 such that CXCR4 activity in the cell is modulated.

16. The nucleic acid of claim 3, wherein the monoclonal antibody or
antigen-binding portion thereof is an IgG1 or IgG4 antibody or a portion
thereof.

17. The nucleic acid of claim 3, wherein the antigen-binding portion of
the monoclonal antibody is a Fab, Fab' or F(ab')2, Fv, dAb, or scFv
fragment.

18. An expression vector comprising the nucleic acid of claim 3.

19. A host cell comprising the expression vector of claim 18.

20. A method for preparing an anti-CXCR4 antibody or an antigen-binding
portion thereof which comprises expressing the antibody or
antigen-binding portion thereof in the host cell of claim 19 and
isolating the antibody or antigen-binding portion thereof from the host
cell.

Description:

BACKGROUND

[0001] Chemokines are a family of about 50 small proteins that modulate
cell trafficking and angiogenesis and also play a significant role in the
tumor microenvironment (Vicari, A. P. and Caux, C. (2002) Cytokine Growth
Factor Rev. 13:143-154). Depending on their structure, chemokines are
classified as C-C chemokines (containing a cysteine-cysteine motif) or
C-X-C chemokines (containing a cysteine-X-cysteine motif). Receptors that
bind such chemokines thus are classified as members of the CCR family or
CXCR family, respectively. One member of the CXCR family is CXCR4, a
seven transmembrane G-protein coupled receptor that is predominantly
expressed on lymphocytes and that activates chemotaxis. CXCR4 binds the
chemokine CXCL12 (SDF-1).

[0002] CXCR4 plays a role in embryogenesis, homeostasis and inflammation.
Studies with mice engineered to be deficient in CXCR4 or SDF-1 implicate
the CXCR4/SDF-1 pathway in organ vascularization, as well as in the
immune and hematopoietic systems (Tachibana, K. et al. (1998) Nature
393:591-594). Moreover, CXCR4 has been shown to function as a coreceptor
for T lymphotrophic HIV-1 isolates (Feng, Y. et al. (1996) Science
272:872-877). CXCR4 also has been shown to be expressed on a wide variety
of cancer cell types. Additionally, the CXCR4/SDF-1 pathway has been
shown to be involved in stimulating the metastatic process in many
different neoplasms (Murphy, P. M. (2001) N. Engl. J. Med. 345:833-835).
For example, CXCR4 and SDF-1 have been shown to mediate organ-specific
metastasis by creating a chemotactic gradient between the primary tumor
site and the metastatic site (Muller, A. et al. (2001) Nature 410:50-56;
Murakami, T. et al. (2002) Cancer Res. 62:7328-7334; Hanahan, D. et al.
(2003) Cancer Res. 63:3005-3008).

SUMMARY

[0003] The present disclosure provides isolated monoclonal antibodies, in
particular human monoclonal antibodies, that bind to human CXCR4 and that
exhibit numerous desirable properties. These properties include the
ability to bind to native human CXCR4 expressed on a cell surface, the
ability to inhibit SDF-1 binding to human CXCR4, the ability to inhibit
SDF-1-induced calcium flux in cells expressing CXCR4, the ability to
inhibit SDF-1-induced migration of cells expressing CXCR4, the ability to
inhibit capillary tube formation by human umbilical vein endothelial
cells (HuVECs), the ability to induce apoptosis in cells expressing
CXCR4, the ability to inhibit tumor cell proliferation in vitro, the
ability to inhibit tumor cell proliferation in vivo, the ability to
inhibit metastases of CXCR4.sup.+ tumor cells and/or the ability to
increase survival time of a CXCR4.sup.+ tumor-bearing subject.

[0004] In one aspect, the instant disclosure pertains to an isolated human
monoclonal antibody, or an antigen-binding portion thereof, wherein the
antibody binds to native human CXCR4 expressed on a cell surface. In one
embodiment, the antibody also inhibits binding of SDF-1 to human CXCR4,
preferably with an EC50 for inhibition of 50 nM or less, or 30 nM or
less, or 15 nM or less, or 10 nM or less, or 5 nM or less, or 3 nM or
less (e.g., an EC50 for inhibition of 28.60 nM or less, or 12.51 nM
or less, or 2.256 nM or less). In another embodiment, the antibody binds
to native human CXCR4 expressed on a cell surface but does not inhibit
binding of SDF-1 to human CXCR4. In yet other embodiments, the antibody
also inhibits SDF-1-induced calcium flux in cells expressing human CXCR4,
preferably with an EC50 for inhibition of 3 nM or less, or 2 nM or
less, or 1 nM or less, or 0.9 nM or less, or 0.8 nM or less, or 0.7 nM or
less, or 0.6 nM or less, or 0.5 nM or less, or 0.4 nM or less (e.g.,
0.9046 nM or less, 0.5684 or less, or 0.3219 nM or less). In yet other
embodiments, the antibody also inhibits SDF-1-induced migration of cells
expressing human CXCR4, preferably with an EC50 for inhibition of 50
nM or less, or 30 nM or less, or 20 nM or less, or 15 nM or less (e.g.,
18.99 nM or less, or 12.44 or less). In still other embodiments, the
antibody also inhibits capillary tube formation by HuVECs, induces
apoptosis of cells expressing CXCR4, inhibits tumor cell proliferation in
vitro, inhibits tumor cell proliferation or induces tumor cell apoptosis
in vivo, inhibits metastases of CXCR4.sup.+ tumor cells and/or increases
survival time of a CXCR4.sup.+ tumor-bearing subject.

[0005] Preferably, the antibody binds to human CXCR4 with high affinity,
such as with a KD of 1×10-7 M or less or with a KD
of 5×10-8 M or less. Preferably, the antibodies of this
disclosure are full-length antibodies (i.e., comprising variable and
constant regions). Furthermore, the antibodies of this disclosure
preferably are raised against full-length human CXCR-4 expressed in its
native conformation on a host cell or in an artificial membrane.

[0006] In a preferred aspect, this disclosure pertains to an isolated
human monoclonal antibody, or an antigen-binding portion thereof, wherein
the antibody:

[0012] In another aspect, this disclosure pertains to an isolated human
monoclonal antibody, or antigen binding portion thereof, wherein the
antibody cross-competes for binding to CXCR4 with a reference antibody,
wherein the reference antibody comprises:

[0017] In certain embodiments, this disclosure provides an isolated
monoclonal antibody, or an antigen-binding portion thereof, comprising a
heavy chain variable region that is the product of or derived from a
human VH 3-48 gene, wherein the antibody specifically binds human
CXCR4. In other embodiments, this disclosure provides an isolated
monoclonal antibody, or an antigen-binding portion thereof, comprising a
light chain variable region that is the product of or derived from a
human VK L15 gene, wherein the antibody specifically binds human
CXCR4. In yet other embodiments, this disclosure provides an isolated
monoclonal antibody, or an antigen-binding portion thereof, comprising a
heavy chain variable region that is the product of or derived from a
human VH 3-48 gene and a light chain variable region that is the
product of or derived from a human VK L15 gene, wherein the antibody
specifically binds human CXCR4.

[0057] In another aspect of this disclosure, antibodies, or
antigen-binding portions thereof, are provided that compete for binding
to CXCR4 with any of the aforementioned antibodies.

[0058] The antibodies of this disclosure can be, for example, full-length
antibodies, for example of an IgG1 or IgG4 isotype. Alternatively, the
antibodies can be antibody fragments, such as Fab, Fab' or Fab'2
fragments, or single chain antibodies.

[0059] This disclosure also provides an immunoconjugate comprising an
antibody of this disclosure, or antigen-binding portion thereof, linked
to a therapeutic agent, such as a cytotoxin or a radioactive isotope.
This disclosure also provides a bispecific molecule comprising an
antibody, or antigen-binding portion thereof, of this disclosure, linked
to a second functional moiety having a different binding specificity than
said antibody, or antigen binding portion thereof.

[0060] Compositions comprising an antibody, or antigen-binding portion
thereof, or immunoconjugate or bispecific molecule of this disclosure and
a pharmaceutically acceptable carrier are also provided.

[0061] Nucleic acid molecules encoding the antibodies, or antigen-binding
portions thereof, of this disclosure are also encompassed by this
disclosure, as well as expression vectors comprising such nucleic acids
and host cells comprising such expression vectors. Methods for preparing
anti-CXCR4 antibodies using the host cells comprising such expression
vectors are also provided and may include the steps of (i) expressing the
antibody in the host cell and (ii) isolating the antibody from the host
cell.

[0062] Another aspect of this disclosure pertains to methods of modulating
CXCR4 activity in a cell, wherein the cells are contacted with an
antibody, or antigen-binding portion thereof, of this disclosure. The
cells can be contacted in vitro by culturing the cells with the antibody
or the cells can be contacted in vivo by administering the antibody to a
subject. In a preferred embodiment, the cells are tumor cells expressing
CXCR4 and the method results in inhibition of the growth of tumor cells
and/or inhibition of metastasis of the tumor cells. In another
embodiment, the cells are T cells expressing CXCR4 and the method results
in inhibition of entry of HIV into the cells. In yet another embodiment,
the cells are lymphocytes in an inflammatory disorder and the methods
result in inhibition of inflammation. In yet another embodiment, the
cells are involved in vascularization and the method results in
modulation of angiogenesis.

[0063] In another aspect, this disclosure pertains to a method of
stimulating mobilization of CD34.sup.+ stem cells from bone marrow to
peripheral blood in a subject, the method comprising administering to the
subject an antibody, or antigen-binding portion thereof, of this
disclosure such that mobilization of CD34.sup.+ stem cells from bone
marrow to peripheral blood is stimulated. The method can further comprise
collecting the CD34.sup.+ stem cells from the peripheral blood, such as
for use in autologous stem cell transplantation.

[0064] Other features and advantages of the instant disclosure will be
apparent from the following detailed description and examples, which
should not be construed as limiting. The contents of all references,
Genbank entries, patents and published patent applications cited
throughout this application are expressly incorporated herein by
reference.

[0090] The present dislcosure relates to isolated monoclonal antibodies,
particularly human monoclonal antibodies, which bind specifically to
native human CXCR4 expressed on a cell surface. In certain embodiments,
the antibodies of this disclosure are derived from particular heavy and
light chain germline sequences and/or comprise particular structural
features such as CDR regions comprising particular amino acid sequences.
This disclosure provides isolated antibodies, methods of making such
antibodies, immunoconjugates and bispecific molecules comprising such
antibodies and pharmaceutical compositions containing the antibodies,
immunoconjugates or bispecific molecules of this disclosure. This
disclosure also relates to methods of using the antibodies, such as to
detect CXCR4, as well as to modulate CXCR4 activity in diseases or
disorders associated with expression of CXCR4 or involving the
CXCR4/SDF-1 pathway, such as cancers, tumor metastasis, HIV infection,
inflammation and angiogenesis. Accordingly, this disclosure also provides
methods of using the anti-CXCR4 antibodies of this disclosure to treat
cancer, for example, to treat a cancer such as breast, ovarian, prostate,
non-small cell lung, pancreatic, thyroid, melanoma, nasopharyngeal, renal
cell, lymphoma, neuroblastoma, glioblastoma, rhabdomyosarcoma,
colorectal, kidney, osteosarcoma, acute lymphoblastic leukemia or acute
myeloid leukemia. Additionally, this disclosure provides methods of using
the anti-CXCR4 antibodies of this disclosure to inhibit tumor metastasis.

[0091] In order that the present disclosure may be more readily
understood, certain terms are first defined. Additional definitions are
set forth throughout the detailed description.

[0092] The term "CXCR4" includes variants, isoforms, homologs, orthologs
and paralogs. For example, antibodies specific for CXCR4 may, in certain
cases, cross-react with CXCR4 from species other than human. In other
embodiments, the antibodies specific for human CXCR4 may be completely
specific for human CXCR4 and may not exhibit species or other types of
cross-reactivity. The term "human CXCR4" refers to human sequence CXCR4,
such as the complete amino acid sequence of human CXCR4 having Genbank
accession number P61073 (SEQ ID NO.: 51). CXCR4 is also known in the art
as, for example, LESTR, Fusin or CD184. The human CXCR4 sequence may
differ from human CXCR4 of SEQ ID NO.: 51 by having, for example,
conserved mutations or mutations in non-conserved regions and the CXCR4
has substantially the same biological function as the human CXCR4 of SEQ
ID NO.: 51. For example, a biological function of human CXCR4 is having
an epitope in the extracellular domain of CXCR4 that is specifically
bound by an antibody of the instant disclosure or the biological function
of human CXCR4 is chemokine binding or involvement in the metastatic
process.

[0093] A particular human CXCR4 sequence will generally be at least 90%
identical in amino acids sequence to human CXCR4 of SEQ ID NO.: 51 and
contains amino acid residues that identify the amino acid sequence as
being human when compared to CXCR4 amino acid sequences of other species
(e.g., murine). In certain cases, a human CXCR4 may be at least 95%, or
even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to
CXCR4 of SEQ ID NO.: 51. In certain embodiments, a human CXCR4 sequence
will display no more than 10 amino acid differences from the CXCR4 of SEQ
ID NO.: 51. In certain embodiments, the human CXCR4 may display no more
than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the
CXCR4 of SEQ ID NO.: 51. Percent identity can be determined as described
herein.

[0094] The term "SDF-1" refers to stromal cell-derived factor 1, which is
a ligand for CXCR4. The term "SDF-1" encompasses different isoforms of
SDF-1, such as SDF-1α and SDF-1β. The amino acid sequence of
human SDF-1α has Genbank accession number NP--954637. The
amino acid sequence of human SDF-1β has Genbank accession number
NP--000600. Human SDF-1 is also described in U.S. Pat. No.
5,756,084. SDF-1 is also known as CXCL12. The amino acid sequence of
human SDF-1 can differ from the SDF-1 of NP--954637 or
NP--000600, as described herein for CXCR4.

[0095] The term "immune response" refers to the action of, for example,
lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,
and soluble macromolecules produced by the above cells or the liver
(including antibodies, cytokines, and complement) that results in
selective damage to, destruction of, or elimination from the human body
of invading pathogens, cells or tissues infected with pathogens,
cancerous cells, or, in cases of autoimmunity or pathological
inflammation, normal human cells or tissues.

[0096] A "signal transduction pathway" refers to the biochemical
relationship between a variety of signal transduction molecules that play
a role in the transmission of a signal from one portion of a cell to
another portion of a cell. As used herein, the phrase "cell surface
receptor" includes, for example, molecules and complexes of molecules
capable of receiving a signal and the transmission of such a signal
across the plasma membrane of a cell. An example of a "cell surface
receptor" of the present disclosure is the CXCR4 receptor.

[0097] The term "antibody" as referred to herein includes whole antibodies
and any antigen binding fragment (i.e., "antigen-binding portion") or
single chains thereof. An "antibody" refers to a glycoprotein comprising
at least two heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds, or an antigen binding portion thereof. Each heavy chain
is comprised of a heavy chain variable region (abbreviated herein as VH)
and a heavy chain constant region. The heavy chain constant region is
comprised of three domains, CH1, CH2 and CH3. Each light
chain is comprised of a light chain variable region (abbreviated herein
as VL) and a light chain constant region. The light chain constant
region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR). Each
VH and VL is composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1, CDR1,
FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light
chains contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells of the
immune system (e.g., effector cells) and the first component (Clq) of the
classical complement system.

[0098] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more fragments of
an antibody that retain the ability to specifically bind to an antigen
(e.g., CXCR4). It has been shown that the antigen-binding function of an
antibody can be performed by fragments of a full-length antibody.
Examples of binding fragments encompassed within the term
"antigen-binding portion" of an antibody include (i) a Fab fragment, a
monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the hinge
region; (iii) a Fab' fragment, which is essentially an Fab with part of
the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd ed.
1993); (iv) a Fd fragment consisting of the VH and CH1 domains;
(v) a Fv fragment consisting of the VL and VH domains of a
single arm of an antibody, (vi) a dAb fragment (Ward et al., (1989)
Nature 341:544-546), which consists of a VH domain; (vii) an
isolated complementarity determining region (CDR); and (viii) a nanobody,
a heavy chain variable region containing a single variable domain and two
constant domains. Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can
be joined, using recombinant methods, by a synthetic linker that enables
them to be made as a single protein chain in which the VL and
VH regions pair to form monovalent molecules (known as single chain
Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston
et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding portion" of an antibody. These antibody fragments are
obtained using conventional techniques known to those with skill in the
art, and the fragments are screened for utility in the same manner as are
intact antibodies.

[0099] An "isolated antibody", as used herein, is intended to refer to an
antibody that is substantially free of other antibodies having different
antigenic specificities (e.g., an isolated antibody that specifically
binds CXCR4 is substantially free of antibodies that specifically bind
antigens other than CXCR4). An isolated antibody that specifically binds
CXCR4 may, however, have cross-reactivity to other antigens, such as
CXCR4 molecules from other species. Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals.

[0100] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody molecules
of single molecular composition. A monoclonal antibody composition
displays a single binding specificity and affinity for a particular
epitope.

[0101] The term "human antibody", as used herein, is intended to include
antibodies having variable regions in which both the framework and CDR
regions are derived from human germline immunoglobulin sequences.
Furthermore, if the antibody contains a constant region, the constant
region also is derived from human germline immunoglobulin sequences. The
human antibodies of this disclosure may include amino acid residues not
encoded by human germline immunoglobulin sequences (e.g., mutations
introduced by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo). However, the term "human antibody", as used herein, is
not intended to include antibodies in which CDR sequences derived from
the germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.

[0102] The term "human monoclonal antibody" refers to antibodies
displaying a single binding specificity, which have variable regions in
which both the framework and CDR regions are derived from human germline
immunoglobulin sequences. In one embodiment, the human monoclonal
antibodies are produced by a hybridoma which includes a B cell obtained
from a transgenic nonhuman animal, e.g., a transgenic mouse, having a
genome comprising a human heavy chain transgene and a light chain
transgene fused to an immortalized cell.

[0103] The term "recombinant human antibody", as used herein, includes all
human antibodies that are prepared, expressed, created or isolated by
recombinant means, such as (a) antibodies isolated from an animal (e.g.,
a mouse) that is transgenic or transchromosomal for human immunoglobulin
genes or a hybridoma prepared therefrom (described further below), (b)
antibodies isolated from a host cell transformed to express the human
antibody, e.g., from a transfectoma, (c) antibodies isolated from a
recombinant, combinatorial human antibody library, and (d) antibodies
prepared, expressed, created or isolated by any other means that involve
splicing of human immunoglobulin gene sequences to other DNA sequences.
Such recombinant human antibodies have variable regions in which the
framework and CDR regions are derived from human germline immunoglobulin
sequences. In certain embodiments, however, such recombinant human
antibodies can be subjected to in vitro mutagenesis (or, when an animal
transgenic for human Ig sequences is used, in vivo somatic mutagenesis)
and thus the amino acid sequences of the VH and VL regions of
the recombinant antibodies are sequences that, while derived from and
related to human germline VH and VL sequences, may not
naturally exist within the human antibody germline repertoire in vivo.

[0104] As used herein, "isotype" refers to the antibody class (e.g., IgM
or IgG1) that is encoded by the heavy chain constant region genes.

[0105] The phrases "an antibody recognizing an antigen" and "an antibody
specific for an antigen" are used interchangeably herein with the term
"an antibody which binds specifically to an antigen."

[0106] The term "human antibody derivatives" refers to any modified form
of the human antibody, e.g., a conjugate of the antibody and another
agent or antibody.

[0107] The term "humanized antibody" is intended to refer to antibodies in
which CDR sequences derived from the germline of another mammalian
species, such as a mouse, have been grafted onto human framework
sequences. Additional framework region modifications may be made within
the human framework sequences.

[0108] The term "chimeric antibody" is intended to refer to antibodies in
which the variable region sequences are derived from one species and the
constant region sequences are derived from another species, such as an
antibody in which the variable region sequences are derived from a mouse
antibody and the constant region sequences are derived from a human
antibody.

[0109] As used herein, an antibody that "specifically binds to human
CXCR4" is intended to refer to an antibody that binds to human CXCR4 (and
possibly CXCR4 from one or more non-human species) but does not
substantially bind to non-CXCR4 proteins. In certain embodiments, an
antibody of the instant disclosure specifically binds to human CXCR4 of
SEQ ID NO.: 51 or a variant thereof. Preferably, the antibody binds to
human CXCR4 with a KD of 1×10-7 M or less, more
preferably 5×10-8 M or less, more preferably 3×10-8
M or less, more preferably 1×10-8 M or less, even more
preferably 5×10-9 M or less.

[0110] The term "does not substantially bind" to a protein or cells, as
used herein, means does not bind or does not bind with a high affinity to
the protein or cells, i.e. binds to the protein or cells with a KD
of 1×10-6 M or more, more preferably 1×10-5 M or
more, more preferably 1×10-4 M or more, more preferably
1×10-3 M or more, even more preferably 1×10-2 M or
more.

[0111] The term "Kassoc" or "Ka", as used herein, is intended to
refer to the association rate of a particular antibody-antigen
interaction, whereas the term "Kdis" or "Kd," as used herein,
is intended to refer to the dissociation rate of a particular
antibody-antigen interaction. The term "KD", as used herein, is
intended to refer to the dissociation constant, which is obtained from
the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed
as a molar concentration (M). KD values for antibodies can be
determined using methods well established in the art. A preferred method
for determining the KD of an antibody is by using surface plasmon
resonance, preferably using a biosensor system such as a Biacore®
system.

[0112] As used herein, the term "high affinity" for an IgG antibody refers
to an antibody having a KD of 1×10-7 M or less, more
preferably 5×10-8 M or less, even more preferably
1×10-8 M or less, even more preferably 5×10-9 M or
less and even more preferably 1×10-9 M or less for a target
antigen. However, "high affinity" binding can vary for other antibody
isotypes. For example, "high affinity" binding for an IgM isotype refers
to an antibody having a KD of 10-6 M or less, more preferably
10-7 M or less, even more preferably 10-8 M or less.

[0113] As used herein, the term "subject" includes any human or nonhuman
animal. The term "nonhuman animal" includes all vertebrates, e.g.,
mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,
horses, cows, chickens, amphibians, reptiles, etc.

[0114] Various aspects of this disclosure are described in further detail
in the following subsections.

Anti-CXCR4 Antibodies

[0115] The antibodies of this disclosure are characterized by particular
functional features or properties of the antibodies. For example, the
antibodies bind to native human CXCR4 expressed on a cell surface.
Preferably, an antibody of this disclosure binds to CXCR4 with high
affinity, for example with a KD of 1×10-7 M or less. The
anti-CXCR4 antibodies of this disclosure preferably exhibit one or more
of the following characteristics:

[0126] (k) increasing survival time of a
CXCR4.sup.+ tumor-bearing subject.

[0127] In certain embodiments, an antibody of this disclosure binds to
native human CXCR4 on a cell surface but does not inhibit binding of
SDF-1 to CXCR4 and does not inhibit SDF-1-induced calcium flux in cells
expressing CXCR4 and does not inhibit SDF-1-induced migration of cells
expressing CXCR4. In other embodiments, an antibody of this disclosure
binds to native human CXCR4 on a cell surface and does inhibit binding of
SDF-1 to CXCR4 and does inhibit SDF-1-induced calcium flux in cells
expressing CXCR4 but does not inhibit SDF-1-induced migration of cells
expressing CXCR4. In still other embodiments, an antibody of this
disclosure binds to native human CXCR4 on a cell surface and does inhibit
binding of SDF-1 to CXCR4 and does inhibit SDF-1-induced calcium flux in
cells expressing CXCR4 and does inhibit SDF-1-induced migration of cells
expressing CXCR4. In still other embodiments, an antibody of this
disclosure binds to native human CXCR4 on a cell surface, does inhibit
binding of SDF-1 to CXCR4, does inhibit SDF-1-induced calcium flux in
cells expressing CXCR4, does inhibit SDF-1-induced migration of cells
expressing CXCR4 and does inhibit capillary tube formation by HuVECs.

[0128] Preferably, an antibody of this disclosure binds to human CXCR4
with a KD of 5×10-8 M or less, binds to human CXCR4 with
a KD of 2×10-8 M or less, binds to human CXCR4 with a
KD of 5×10-9 M or less, binds to human CXCR4 with a
KD of 4×10-9 M or less, binds to human CXCR4 with a
KD of 3×10-9 M or less, or binds to human CXCR4 with a
KD of 2×10-9 M or less.

[0129] Preferably, an antibody of the inhibits binding of SDF-1 to human
CXCR4 with an EC50 for inhibition of 50 nM or less, more preferably
30 nM or less, or 15 nM or less, or 10 nM or less, or 5 nM or less, or 3
nM or less (e.g., an EC50 for inhibition of 28.60 nM or less, or
12.51 nM or less, or 2.256 nM or less)

[0130] Preferably, an antibody of this disclosure inhibits SDF-1-induced
calcium flux in cells expressing human CXCR4 with an EC50 for
inhibition of 3 nM or less, more preferably 2 nM or less, or 1 nM or
less, or 0.9 nM or less, or 0.8 nM or less, or 0.7 nM or less, or 0.6 nM
or less, or 0.5 nM or less, or 0.4 nM or less (e.g., 0.9046 nM or less,
0.5684 or less, or 0.3219 nM or less).

[0131] Preferably, an antibody of this disclosure inhibits SDF-1-induced
migration of cells expressing human CXCR4 with an EC50 for
inhibition of 50 nM or less, more preferably 30 nM or less, or 20 nM or
less, or 15 nM or less (e.g., 18.99 nM or less, or 12.44 or less).

[0132] Standard assays to evaluate the binding ability of the antibodies
toward native human CXCR4 expressed on a cell surface are known in the
art, including for example, flow cytometry analysis using a cell line
that naturally expresses native CXCR4 or that has been transfected to
express native CXCR4. Suitable assays are described in detail in the
Examples. A preferred cell line that expresses native CXCR4 is the CEM T
cell line. Suitable assays for evaluating inhibition of binding of SDF-1,
inhibition of SDF-1 induced calcium flux, inhibition of SDF-1 induced
cell migration, inhibition of capillary tube formation by HuVECs,
induction of apoptosis in cells expressing CXCR4 in vitro and/or in vivo,
inhibition of growth of CXCR4.sup.+ tumor cells in vitro and/or in vivo,
and/or inhibition of metastases of CXCR4.sup.+ tumor cells are also
described in detail in the Examples. Binding affinity of the antibodies
also can be determined by standard methods, such as by Scatchard
analysis.

[0133] Monoclonal Antibodies F7, F9, D1 and E2

[0134] Preferred antibodies of this disclosure are the human monoclonal
antibodies F7, F9, D1 and E2, isolated and structurally characterized as
described in Examples 1 and 2. The VH amino acid sequences of F7,
F9, D1 and E2 are shown in SEQ ID NOs: 25, 26, 27 and 28, respectively.
The VL amino acid sequences of F7, F9, D1 and E2 are shown in SEQ ID
NOs: 29, 30, 31 and 32, respectively. Additionally, alternative forms of
F7, F9, D1 and E2, in which certain framework residues were substituted
with a germline residue, were created and are referred to herein as F7GL,
F9GL, D1GL and E2GL. The VH amino acid sequences of F7GL, F9GL, D1GL
and E2GL are shown in SEQ ID NOs: 41, 42, 43 and 44, respectively. The
VL amino acid sequences of F7GL, F9GL, D1GL and E2GL are shown in
SEQ ID NOs: 45, 46, 47 and 48, respectively.

[0135] Given that each of these antibodies can bind to CXCR4, the VH
and VL sequences can be "mixed and matched" to create other
anti-CXCR4 binding molecules of this disclosure. CXCR4 binding of such
"mixed and matched" antibodies can be tested using the binding assays
described above and in the Examples (e.g., flow cytometry with CEM
cells). Preferably, when VH and VL chains are mixed and
matched, a VH sequence from a particular VH/VL pairing is
replaced with a structurally similar VH sequence. Likewise,
preferably a VL sequence from a particular VH/VL pairing
is replaced with a structurally similar VL sequence.

[0143] In another aspect, this disclosure provides antibodies that
comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of F7,
F9, D1 or E2, or combinations thereof. The amino acid sequences of the
VH CDR1s of F7, F9, D1 and E2 are shown in SEQ ID NOs: 1-4,
respectively. The amino acid sequences of the VH CDR2s of F7, F9, D1
and E2 are shown in SEQ ID NOs: 5-8, respectively. The amino acid
sequences of the VH CDR3s of F7, F9, D1 and E2 are shown in SEQ ID
NOs: 9-12, respectively. The amino acid sequences of the Vk CDR1s of
F7, F9, D1 and E2 are shown in SEQ ID NOs: 13-16, respectively. The amino
acid sequences of the Vk CDR2s of F7, F9, D1 and E2 are shown in SEQ
ID NOs: 17-20, respectively. The amino acid sequences of the Vk CDR3
s of F7, F9, D1 and E2 are shown in SEQ ID NOs: 21-24, respectively. The
CDR regions are delineated using the Kabat system (Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242).

[0144] Given that each of these antibodies can bind to CXCR4 and that
antigen-binding specificity is provided primarily by the CDR1, CDR2, and
CDR3 regions, the VH CDR1, CDR2, and CDR3 sequences and Vk
CDR1, CDR2, and CDR3 sequences can be "mixed and matched" (i.e., CDRs
from different antibodies can be mixed and match, although each antibody
must contain a VH CDR1, CDR2, and CDR3 and a Vk CDR1, CDR2, and
CDR3) to create other anti-CXCR4 binding molecules of this disclosure.
CXCR4 binding of such "mixed and matched" antibodies can be tested using
the binding assays described above and in the Examples (e.g., ELISAs,
Biacore® analysis). Preferably, when VH CDR sequences are mixed
and matched, the CDR1, CDR2 and/or CDR3 sequence from a particular
VH sequence is replaced with a structurally similar CDR sequence(s).
Likewise, when Vk CDR sequences are mixed and matched, the CDR1,
CDR2 and/or CDR3 sequence from a particular Vk sequence preferably
is replaced with a structurally similar CDR sequence(s). It will be
readily apparent to the ordinarily skilled artisan that novel VH and
VL sequences can be created by substituting one or more VH
and/or VL CDR region sequences with structurally similar sequences
from the CDR sequences disclosed herein for monoclonal antibodies
antibodies F7, F9, D1 and E2.

[0178] It is well known in the art that the CDR3 domain, independently
from the CDR1 and/or CDR2 domain(s), alone can determine the binding
specificity of an antibody for a cognate antigen and that multiple
antibodies can predictably be generated having the same binding
specificity based on a common CDR3 sequence. See, for example, Klimka et
al., British J. of Cancer 83(2):252-260 (2000) (describing the production
of a humanized anti-CD30 antibody using only the heavy chain variable
domain CDR3 of murine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol.
Biol. 296:833-849 (2000) (describing recombinant epithelial
glycoprotein-2 (EGP-2) antibodies using only the heavy chain CDR3
sequence of the parental murine MOC-31 anti-EGP-2 antibody); Rader et
al., Proc. Natl. Acad. Sci. U.S.A. 95:8910-8915 (1998) (describing a
panel of humanized anti-integrin αvβ3 antibodies
using a heavy and light chain variable CDR3 domain of a murine
anti-integrin αvβ3 antibody LM609 wherein each
member antibody comprises a distinct sequence outside the CDR3 domain and
capable of binding the same epitope as the parent muring antibody with
affinities as high or higher than the parent murine antibody); Barbas et
al., J. Am. Chem. Soc. 116:2161-2162 (1994) (disclosing that the CDR3
domain provides the most significant contribution to antigen binding);
Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533 (1995)
(describing the grafting of heavy chain CDR3 seqeunces of three Fabs
(SI-1, SI-40, and SI-32) against human placental DNA onto the heavy chain
of an anti-tetanus toxoid Fab thereby replacing the existing heavy chain
CDR3 and demonstrating that the CDR3 domain alone conferred binding
specificity); Ditzel et al., J. Immunol. 157:739-749 (1996) (describing
grafting studies wherein transfer of only the heavy chain CDR3 of a
parent polyspecific Fab LNA3 to a heavy chain of a monospecific IgG
tetanus toxoid-binding Fab p313 antibody was sufficient to retain binding
specificity of the parent Fab); Berezov et al., BIAjournal 8: Scientific
Review 8 (2001) (describing peptide mimetics based on the CDR3 of an
anti-HER2 monoclonal antibody; Igarashi et al., J. Biochem (Tokyo)
117:452-7 (1995) (describing a 12 amino acid synthetic polypeptide
corresponding to the CDR3 domain of an anti-phosphatidylserine antibody);
Bourgeois et al., J. Virol 72:807-10 (1998) (showing that a signle petide
derived form the heavy chain CDR3 domain of an anti-respiratory syncytial
virus (RSV) antibody was capable of neutralizing the virus in vitro);
Levi et al., Proc. Natl. Acad. Sci. U.S.A. 90:4374-8 (1993) (describing a
peptide based on the heavy chain CDR3 domain of a murine anti-HIV
antibody); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994)
(describing enabling binding of an scFv by grafting the heavy chain CDR3
region of a Z-DNA-binding antibody) and Xu and Davis, Immunity 13:37-45
(2000) (describing that diversity at the heavy chain CDR3 is sufficient
to permit otherwise idential IgM molecules to distinguish between a
variety of hapten and protein antigens). See also, U.S. Pat. Nos.
6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925;
5,833,943; 5,762,905 and 5,760,185, describing patented antibodies
defined by a single CDR domain. Each of these references is hereby
incorporated by reference in its entirety.

[0179] Accordingly, the present disclosure provides monoclonal antibodies
comprising one or more heavy and/or light chain CDR3 domains from an
antibody derived from a human or non-human animal, wherein the monoclonal
antibody is capable of specifically binding to CXCR4. Within certain
aspects, the present disclosure provides monoclonal antibodies comprising
one or more heavy and/or light chain CDR3 domain from a non-human
antibody, such as a mouse or rat antibody, wherein the monoclonal
antibody is capable of specifically binding to CXCR4. Within some
embodiments, such inventive antibodies comprising one or more heavy
and/or light chain CDR3 domain from a non-human antibody (a) are capable
of competing for binding with; (b) retain the functional characteristics;
(c) bind to the same epitope; and/or (d) have a similar binding affinity
as the corresponding parental non-human antibody.

[0180] Within other aspects, the present disclosure provides monoclonal
antibodies comprising one or more heavy and/or light chain CDR3 domain
from a human antibody, such as, for example, a human antibody obtained
from a non-human animal, wherein the human antibody is capable of
specifically binding to CXCR4. Within other aspects, the present
disclosure provides monoclonal antibodies comprising one or more heavy
and/or light chain CDR3 domain from a first human antibody, such as, for
example, a human antibody obtained from a non-human animal, wherein the
first human antibody is capable of specifically binding to CXCR4 and
wherein the CDR3 domain from the first human antibody replaces a CDR3
domain in a human antibody that is lacking binding specificity for CXCR4
to generate a second human antibody that is capable of specifically
binding to CXCR4. Within some embodiments, such inventive antibodies
comprising one or more heavy and/or light chain CDR3 domain from the
first human antibody (a) are capable of competing for binding with; (b)
retain the functional characteristics; (c) bind to the same epitope;
and/or (d) have a similar binding affinity as the corresponding parental
first human antibody.

[0182] For example, in a preferred embodiment, this disclosure provides an
isolated monoclonal antibody, or an antigen-binding portion thereof,
comprising a heavy chain variable region that is the product of or
derived from a human VH 3-48 gene, wherein the antibody specifically
binds CXCR4. In another preferred embodiment, this disclosure provides an
isolated monoclonal antibody, or an antigen-binding portion thereof,
comprising a light chain variable region that is the product of or
derived from a human VK L15 gene, wherein the antibody specifically
binds CXCR4. In yet another preferred embodiment, this disclosure
provides an isolated monoclonal antibody, or antigen-binding portion
thereof, wherein the antibody comprises a heavy chain variable region
that is the product of or derived from a human VH 3-48 gene and
comprises a light chain variable region that is the product of or derived
from a human VK L15 gene, wherein the antibody specifically binds to
CXCR4, preferably human CXCR4. Such antibodies also may possess one or
more of the functional characteristics described in detail above, such as
binding to native CXCR4 expressed on a cell surface, inhibition of SDF-1
binding to CXCR4, inhibition of SDF-1-induced calcium flux in cells
expressing CXCR4, inhibition of SDF-1-induced migration of cells
expressing CXCR4, inhibition of capillary tube formation by HuVECs,
induction of apoptosis in cells expressing CXCR4 in vitro and/or in vivo,
inhibition of growth of CXCR4.sup.+ tumor cells in vitro and/or in vivo,
and/or inhibition of metastases of CXCR4.sup.+ tumor cells.

[0183] Examples of antibodies having VH and VK of VH 3-48
and VK L15, respectively, are the F7, F9, D1 and E2 antibodies.

[0184] As used herein, a human antibody comprises heavy or light chain
variable regions that is "the product of" or "derived from" a particular
germline sequence if the variable regions of the antibody are obtained
from a system that uses human germline immunoglobulin genes. Such systems
include immunizing a transgenic mouse carrying human immunoglobulin genes
with the antigen of interest or screening a human immunoglobulin gene
library displayed on phage with the antigen of interest. A human antibody
that is "the product of" or "derived from" a human germline
immunoglobulin sequence can be identified as such by comparing the amino
acid sequence of the human antibody to the amino acid sequences of human
germline immunoglobulins and selecting the human germline immunoglobulin
sequence that is closest in sequence (i.e., greatest % identity) to the
sequence of the human antibody. A human antibody that is "the product of"
or "derived from" a particular human germline immunoglobulin sequence may
contain amino acid differences as compared to the germline sequence, due
to, for example, naturally-occurring somatic mutations or intentional
introduction of site-directed mutation. However, a selected human
antibody typically is at least 90% identical in amino acids sequence to
an amino acid sequence encoded by a human germline immunoglobulin gene
and contains amino acid residues that identify the human antibody as
being human when compared to the germline immunoglobulin amino acid
sequences of other species (e.g., murine germline sequences). In certain
cases, a human antibody may be at least 95%, or even at least 96%, 97%,
98%, or 99% identical in amino acid sequence to the amino acid sequence
encoded by the germline immunoglobulin gene. Typically, a human antibody
derived from a particular human germline sequence will display no more
than 10 amino acid differences from the amino acid sequence encoded by
the human germline immunoglobulin gene. In certain cases, the human
antibody may display no more than 5, or even no more than 4, 3, 2, or 1
amino acid difference from the amino acid sequence encoded by the
germline immunoglobulin gene.

Homologous Antibodies

[0185] In yet another embodiment, an antibody of this disclosure comprises
heavy and light chain variable regions comprising amino acid sequences
that are homologous to the amino acid sequences of the preferred
antibodies described herein, and wherein the antibodies retain the
desired functional properties of the anti-CXCR4 antibodies of this
disclosure.

[0191] In various embodiments, the antibody can be, for example, a human
antibody, a humanized antibody or a chimeric antibody.

[0192] In other embodiments, the VH and/or VL amino acid
sequences may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the
sequences set forth above. An antibody having VH and VL regions
having high (i.e., 80% or greater) homology to the VH and VL
regions of the sequences set forth above, can be obtained by mutagenesis
(e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid
molecules encoding SEQ ID NOs: 25-32 or 41-48, followed by testing of the
encoded altered antibody for retained function (i.e., the functions set
forth above) using the functional assays described herein.

[0193] As used herein, the percent homology between two amino acid
sequences is equivalent to the percent identity between the two
sequences. The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences (i.e., %
homology=# of identical positions/total # of positions×100), taking
into account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity between two
sequences can be accomplished using a mathematical algorithm, as
described in the non-limiting examples below.

[0194] The percent identity between two amino acid sequences can be
determined using the algorithm of E. Meyers and W. Miller (Comput. Appl.
Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN
program (version 2.0), using a PAM120 weight residue table, a gap length
penalty of 12 and a gap penalty of 4. In addition, the percent identity
between two amino acid sequences can be determined using the Needleman
and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been
incorporated into the GAP program in the GCG software package (available
at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6.

[0195] Additionally or alternatively, the protein sequences of the present
disclosure can further be used as a "query sequence" to perform a search
against public databases to, for example, to identify related sequences.
Such searches can be performed using the XBLAST program (version 2.0) of
Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches
can be performed with the XBLAST program, score=50, wordlength=3 to
obtain amino acid sequences homologous to the antibody molecules of this
disclosure. To obtain gapped alignments for comparison purposes, Gapped
BLAST can be utilized as described in Altschul et al., (1997) Nucleic
Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g., XBLAST
and NBLAST) are useful. See www.ncbi.nlm.nih.gov.

[0202] In various embodiments, the antibody can be, for example, human
antibodies, humanized antibodies or chimeric antibodies.

[0203] As used herein, the term "conservative sequence modifications" is
intended to refer to amino acid modifications that do not significantly
affect or alter the binding characteristics of the antibody containing
the amino acid sequence. Such conservative modifications include amino
acid substitutions, additions and deletions. Modifications can be
introduced into an antibody of this disclosure by standard techniques
known in the art, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Conservative amino acid substitutions are ones in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar side
chains have been defined in the art. These families include amino acids
with basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine), beta-branched
side chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within the CDR regions of an antibody of this
disclosure can be replaced with other amino acid residues from the same
side chain family and the altered antibody can be tested for retained
function (i.e., the functions set forth above) using the functional
assays described herein.

Antibodies that Bind to the Same Epitope as Anti-CXCR4 Antibodies

[0204] In another embodiment, this disclosure provides antibodies that
bind to the same epitope on human CXCR4 as any of the anti-CXCR4
monoclonal antibodies of this disclosure (i.e., antibodies that have the
ability to cross-compete for binding to CXCR4 with any of the monoclonal
antibodies of this disclosure). In preferred embodiments, the reference
antibody for cross-competition studies can be the monoclonal antibody F7
(having VH and VL sequences as shown in SEQ ID NOs: 25 and 29,
respectively), or the monoclonal antibody F9 (having VH and VL
sequences as shown in SEQ ID NOs: 26 and 30, respectively) or the
monoclonal antibody D1 (having VH and VL sequences as shown in
SEQ ID NOs: 27 and 31, respectively) or the monoclonal antibody E2
(having VH and VL sequences as shown in SEQ ID NOs: 28 and 32,
respectively).

[0205] Such cross-competing antibodies can be identified based on their
ability to cross-compete with F7, F9, D1 or E2 in standard CXCR4 binding
assays. For example, flow cytometry with CEM cells may be used to
demonstrate cross-competition with the antibodies of the current
disclosure, wherein the reference antibody is labeled with FITC and the
ability of a test antibody to inhibit the binding of the FITC-labeled
reference antibody to CEM cells is evaluated. The ability of a test
antibody to inhibit the binding of, for example, F7, F9, D1 or E2, to
human CXCR4 demonstrates that the test antibody can compete with F7, F9,
D1 or E2 for binding to human CXCR4 and thus binds to the same epitope on
human CXCR4 as F7, F9, D1 or E2. In a preferred embodiment, the antibody
that binds to the same epitope on CXCR4 as F7, F9, D1 or E2 is a human
monoclonal antibody. Such human monoclonal antibodies can be prepared and
isolated as described in the Examples.

Engineered and Modified Antibodies

[0206] An antibody of this disclosure further can be prepared using an
antibody having one or more of the VH and/or VL sequences
disclosed herein as starting material to engineer a modified antibody,
which modified antibody may have altered properties from the starting
antibody. An antibody can be engineered by modifying one or more residues
within one or both variable regions (i.e., VH and/or VL), for
example within one or more CDR regions and/or within one or more
framework regions. Additionally or alternatively, an antibody can be
engineered by modifying residues within the constant region(s), for
example to alter the effector function(s) of the antibody.

[0207] In certain embodiments, CDR grafting can be used to engineer
variable regions of antibodies. Antibodies interact with target antigens
predominantly through amino acid residues that are located in the six
heavy and light chain complementarity determining regions (CDRs). For
this reason, the amino acid sequences within CDRs are more diverse
between individual antibodies than sequences outside of CDRs. Because CDR
sequences are responsible for most antibody-antigen interactions, it is
possible to express recombinant antibodies that mimic the properties of
specific naturally occurring antibodies by constructing expression
vectors that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody with
different properties (see, e.g., Riechmann, L. et al. (1998) Nature
332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al.
(1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.
5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762
and 6,180,370 to Queen et al.)

[0209] Such framework sequences can be obtained from public DNA databases
or published references that include germline antibody gene sequences.
For example, germline DNA sequences for human heavy and light chain
variable region genes can be found in the "VBase" human germline sequence
database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), as
well as in Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health and
Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.
(1992) "The Repertoire of Human Germline VH Sequences Reveals about
Fifty Groups of VH Segments with Different Hypervariable Loops" J.
Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A Directory of
Human Germ-line VH Segments Reveals a Strong Bias in their Usage"
Eur. J. Immunol. 24:827-836; the contents of each of which are expressly
incorporated herein by reference. As another example, the germline DNA
sequences for human heavy and light chain variable region genes can be
found in the Genbank database. For example, the following heavy chain
germline sequences found in the HCo7 HuMAb mouse are available in the
accompanying Genbank accession numbers: 1-69 (NG--0010109,
NT--024637 and BC070333), 3-33 (NG--0010109 and
NT--024637) and 3-7 (NG--0010109 and NT--024637). As
another example, the following heavy chain germline sequences found in
the HCo12 HuMAb mouse are available in the accompanying Genbank accession
numbers: 1-69 (NG--0010109, NT--024637 and BC070333), 5-51
(NG--0010109 and NT--024637), 4-34 (NG--0010109 and
NT--024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678). Yet another
source of human heavy and light chain germline sequences is the database
of human immunoglobulin genes available from IMGT (http://imgt.cines.fr).

[0210] Antibody protein sequences are compared against a compiled protein
sequence database using one of the sequence similarity searching methods
called the Gapped BLAST (Altschul et al. (1997) Nucleic Acids Research
25:3389-3402), which is well known to those skilled in the art. BLAST is
a heuristic algorithm in that a statistically significant alignment
between the antibody sequence and the database sequence is likely to
contain high-scoring segment pairs (HSP) of aligned words. Segment pairs
whose scores cannot be improved by extension or trimming is called a hit.
Briefly, the nucleotide sequences of VBASE origin
(http://vbase.mrc-cpe.cam.ac.uk/vbase1/list2.php) are translated and the
region between and including FR1 through FR3 framework region is
retained. The database sequences have an average length of 98 residues.
Duplicate sequences which are exact matches over the entire length of the
protein are removed. A BLAST search for proteins using the program blastp
with default, standard parameters except the low complexity filter, which
is turned off, and the substitution matrix of BLOSUM62, filters for top 5
hits yielding sequence matches. The nucleotide sequences are translated
in all six frames and the frame with no stop codons in the matching
segment of the database sequence is considered the potential hit. This is
in turn confirmed using the BLAST program tblastx, which translates the
antibody sequence in all six frames and compares those translations to
the VBASE nucleotide sequences dynamically translated in all six frames.
Other human germline sequence databases, such as that available from IMGT
(http://imgt.cines.fr), can be searched similarly to VBASE as described
above.

[0211] The identities are exact amino acid matches between the antibody
sequence and the protein database over the entire length of the sequence.
The positives (identities+substitution match) are not identical but amino
acid substitutions guided by the BLOSUM62 substitution matrix. If the
antibody sequence matches two of the database sequences with same
identity, the hit with most positives would be decided to be the matching
sequence hit.

[0212] Preferred framework sequences for use in the antibodies of this
disclosure are those that are structurally similar to the framework
sequences used by selected antibodies of this disclosure, e.g., similar
to the VH 3-48 framework sequences (SEQ ID NO: 49) and/or the
VK L15 framework sequence (SEQ ID NO: 50) used by preferred
monoclonal antibodies of this disclosure. The VH CDR1, CDR2, and
CDR3 sequences, and the VK CDR1, CDR2, and CDR3 sequences, can be
grafted onto framework regions that have the identical sequence as that
found in the germline immunoglobulin gene from which the framework
sequence derive, or the CDR sequences can be grafted onto framework
regions that contain one or more mutations as compared to the germline
sequences. For example, it has been found that in certain instances it is
beneficial to mutate residues within the framework regions to maintain or
enhance the antigen binding ability of the antibody (see e.g., U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0213] Another type of variable region modification is to mutate amino
acid residues within the VH and/or VK CDR1, CDR2 and/or CDR3
regions to thereby improve one or more binding properties (e.g.,
affinity) of the antibody of interest. Site-directed mutagenesis or
PCR-mediated mutagenesis can be performed to introduce the mutation(s)
and the effect on antibody binding, or other functional property of
interest, can be evaluated in in vitro or in vivo assays as described
herein and provided in the Examples. Preferably conservative
modifications (as discussed above) are introduced. The mutations may be
amino acid substitutions, additions or deletions, but are preferably
substitutions. Moreover, typically no more than one, two, three, four or
five residues within a CDR region are altered.

[0214] Accordingly, in another embodiment, the instant disclosure provides
isolated anti-CXCR4 monoclonal antibodies, or antigen binding portions
thereof, comprising a heavy chain variable region comprising: (a) a
VH CDR1 region comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 1-4, or an amino acid sequence having
one, two, three, four or five amino acid substitutions, deletions or
additions as compared to SEQ ID NOs: 1-4; (b) a VH CDR2 region
comprising an amino acid sequence selected from the group consisting of
SEQ ID NOs: 5-8, or an amino acid sequence having one, two, three, four
or five amino acid substitutions, deletions or additions as compared to
SEQ ID NOs: 5-8; (c) a VH CDR3 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 9-12, or an
amino acid sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs: 9-12;
(d) a VK CDR1 region comprising an amino acid sequence selected from
the group consisting of SEQ ID NOs: 13-16, or an amino acid sequence
having one, two, three, four or five amino acid substitutions, deletions
or additions as compared to SEQ ID NOs: 13-16; (e) a VK CDR2 region
comprising an amino acid sequence selected from the group consisting of
SEQ ID NOs: 17-20, or an amino acid sequence having one, two, three, four
or five amino acid substitutions, deletions or additions as compared to
SEQ ID NOs: 17-20; and (f) a VK CDR3 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 21-24, or an
amino acid sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs: 21-24.

[0215] Engineered antibodies of this disclosure include those in which
modifications have been made to framework residues within VH and/or
VK, e.g. to improve the properties of the antibody. Typically such
framework modifications are made to decrease the immunogenicity of the
antibody. For example, one approach is to "backmutate" one or more
framework residues to the corresponding germline sequence. More
specifically, an antibody that has undergone somatic mutation may contain
framework residues that differ from the germline sequence from which the
antibody is derived. Such residues can be identified by comparing the
antibody framework sequences to the germline sequences from which the
antibody is derived.

[0216] For example, for the F7 VH region, the following framework
region amino acid positions (using the Kabat numbering system) differ
from germline: 1, 6 and 25. One, two or all three of these positions can
be backmutated to germline sequences by making one, two or all three of
the following substitutions: Q1E, Q6E and A25S. A preferred modified form
of the F7 VH region is F7GL VH (the amino acid sequence of
which is shown in FIG. 5A and in SEQ ID NO: 41), which has the following
framework substitutions: Q1E and Q6E.

[0217] Furthermore, for the F7 Vk region, the following framework
region amino acid positions (using the Kabat numbering system) differ
from germline: 1, 3 and 84. One, two or all three of these positions can
be backmutated to germline sequences by making one, two or all three of
the following substitutions: A1D, R3Q and V84A. A preferred modified form
of the F7 Vk region is F7GL Vk (the amino acid sequence of
which is shown in FIG. 5B and in SEQ ID NO: 45), which has the following
framework substitutions: MD and R3Q.

[0218] Furthermore, for the F9 VH region, the following framework
region amino acid positions (using the Kabat numbering system) differ
from germline: 1, 6 and 25. One, two or all three of these positions can
be backmutated to germline sequences by making one, two or all three of
the following substitutions: Q1E, Q6E and A25S. A preferred modified form
of the F9 VH region is F9GL VH (the amino acid sequence of
which is shown in FIG. 6A and in SEQ ID NO: 42), which has the following
framework substitutions: Q1E and Q6E.

[0219] Furthermore, for the F9 Vk region, the following framework
region amino acid positions (using the Kabat numbering system) differ
from germline: 1, 3, 4 and 60. One, two, three or all four of these
positions can be backmutated to germline sequences by making one, two,
three or all four of the following substitutions: E1D, V3Q, L4M and P60S.
A preferred modified form of the F9 Vk region is F9GL Vk (the
amino acid sequence of which is shown in FIG. 6B and in SEQ ID NO: 46),
which has the following framework substitutions: E1D, V3Q and L4M.

[0220] Furthermore, for the D1 VH region, the following framework
region amino acid positions (using the Kabat numbering system) differ
from germline: 6, 25 and 76. One, two or all three of these positions can
be backmutated to germline sequences by making one, two or all three of
the following substitutions: Q6E, A25S and R76K. A preferred modified
form of the D1 VH region is D1GL VH (the amino acid sequence of
which is shown in FIG. 7A and in SEQ ID NO: 43), which has the following
framework substitution: Q6E.

[0221] Furthermore, for the D1 Vk region, the following framework
region amino acid positions (using the Kabat numbering system) differ
from germline: 1, 3, 4, 45 and 46. One, two, three, four or all five of
these positions can be backmutated to germline sequences by making one,
two, three, four or all five of the following substitutions: V1D, W3Q,
V4M, E45K and L46S. A preferred modified form of the D1 Vk region is
D1GL Vk (the amino acid sequence of which is shown in FIG. 7B and in
SEQ ID NO: 47), which has the following framework substitutions: V1D, W3Q
and V4M.

[0222] Furthermore, for the E2 VH region, the following framework
region amino acid positions (using the Kabat numbering system) differ
from germline: 6 and 25. One or both of these positions can be
backmutated to germline sequences by making one or both of the following
substitutions: Q6E and A25S. A preferred modified form of the E2 VH
region is E2GL VH (the amino acid sequence of which is shown in FIG.
8A and in SEQ ID NO: 44), which has the following framework substitution:
Q6E.

[0223] Furthermore, for the E2 Vk region, the following framework
region amino acid positions (using the Kabat numbering system) differ
from germline: 1, 3 and 4. One, two or all three of these positions can
be backmutated to germline sequences by making one, two or all three of
the following substitutions: E1D, V3Q and L4M. A preferred modified form
of the E2 Vk region is E2GL Vk (the amino acid sequence of
which is shown in FIG. 8B and in SEQ ID NO: 48), which has the following
framework substitutions: E1D, V3Q and L4M.

[0224] Another type of framework modification involves mutating one or
more residues within the framework region, or even within one or more CDR
regions, to remove T cell epitopes to thereby reduce the potential
immunogenicity of the antibody. This approach is also referred to as
"deimmunization" and is described in father detail in U.S. Patent
Publication No. 20030153043 by Carr et al.

[0225] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of this disclosure may be engineered
to include modifications within the Fc region, typically to alter one or
more functional properties of the antibody, such as serum half-life,
complement fixation, Fc receptor binding, and/or antigen-dependent
cellular cytotoxicity. Furthermore, an antibody of this disclosure may be
chemically modified (e.g., one or more chemical moieties can be attached
to the antibody) or be modified to alter its glycosylation, again to
alter one or more functional properties of the antibody. Each of these
embodiments is described in further detail below. The numbering of
residues in the Fc region is that of the EU index of Kabat.

[0226] In one embodiment, the hinge region of CH1 is modified such that
the number of cysteine residues in the hinge region is altered, e.g.,
increased or decreased. This approach is described further in U.S. Pat.
No. 5,677,425 by Bodmer et al. The number of cysteine residues in the
hinge region of CH1 is altered to, for example, facilitate assembly of
the light and heavy chains or to increase or decrease the stability of
the antibody.

[0227] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the antibody. More
specifically, one or more amino acid mutations are introduced into the
CH2-CH3 domain interface region of the Fc-hinge fragment such that the
antibody has impaired Staphylococcyl protein A (SpA) binding relative to
native Fc-hinge domain SpA binding. This approach is described in further
detail in U.S. Pat. No. 6,165,745 by Ward et al.

[0228] In another embodiment, the antibody is modified to increase its
biological half life. Various approaches are possible. For example, one
or more of the following mutations can be introduced: T252L, T254S,
T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to
increase the biological half life, the antibody can be altered within the
CH1 or CL region to contain a salvage receptor binding epitope taken from
two loops of a CH2 domain of an Fc region of an IgG, as described in U.S.
Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

[0229] In yet other embodiments, the Fc region is altered by replacing at
least one amino acid residue with a different amino acid residue to alter
the effector function(s) of the antibody. For example, one or more amino
acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320
and 322 can be replaced with a different amino acid residue such that the
antibody has an altered affinity for an effector ligand but retains the
antigen-binding ability of the parent antibody. The effector ligand to
which affinity is altered can be, for example, an Fc receptor or the C1
component of complement. This approach is described in further detail in
U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0230] In another example, one or more amino acids selected from amino
acid residues 329, 331 and 322 can be replaced with a different amino
acid residue such that the antibody has altered Clq binding and/or
reduced or abolished complement dependent cytotoxicity (CDC). This
approach is described in further detail in U.S. Pat. No. 6,194,551 by
Idusogie et al.

[0231] In another example, one or more amino acid residues within amino
acid positions 231 and 239 are altered to thereby alter the ability of
the antibody to fix complement. This approach is described further in PCT
Publication WO 94/29351 by Bodmer et al.

[0233] In still another embodiment, the glycosylation of an antibody is
modified. For example, an aglycoslated antibody can be made (i.e., the
antibody lacks glycosylation). Glycosylation can be altered to, for
example, increase the affinity of the antibody for antigen. Such
carbohydrate modifications can be accomplished by, for example, altering
one or more sites of glycosylation within the antibody sequence. For
example, one or more amino acid substitutions can be made that result in
elimination of one or more variable region framework glycosylation sites
to thereby eliminate glycosylation at that site. Such aglycosylation may
increase the affinity of the antibody for antigen. Such an approach is
described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by
Co et al.

[0234] Additionally or alternatively, an antibody can be made that has an
altered type of glycosylation, such as a hypofucosylated antibody having
reduced amounts of fucosyl residues or an antibody having increased
bisecting GlcNac structures. Such altered glycosylation patterns have
been demonstrated to increase the ADCC ability of antibodies. Such
carbohydrate modifications can be accomplished by, for example,
expressing the antibody in a host cell with altered glycosylation
machinery. Cells with altered glycosylation machinery have been described
in the art and can be used as host cells in which to express recombinant
antibodies of this disclosure to thereby produce an antibody with altered
glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack
the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such
that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack
fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8.sup.-/-
cell lines were created by the targeted disruption of the FUT8 gene in
CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication
No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004)
Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai
et al. describes a cell line with a functionally disrupted FUT8 gene,
which encodes a fucosyl transferase, such that antibodies expressed in
such a cell line exhibit hypofucosylation by reducing or eliminating the
alpha 1,6 bond-related enzyme. Hanai et al. also describe cell lines
which have a low enzyme activity for adding fucose to the
N-acetylglucosamine that binds to the Fc region of the antibody or does
not have the enzyme activity, for example the rat myeloma cell line YB2/0
(ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describes a
variant CHO cell line, Lec13 cells, with reduced ability to attach fucose
to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of
antibodies expressed in that host cell (see also Shields, R. L. et al.
(2002) J. Biol. Chem. 277:26733-26740). Antibodies with a modified
glycosylation profile can also be produced in chicken eggs, as described
in US Patent Application No. PCT/US06/05853. Alternatively, antibodies
with a modified glycosylation profile can be produced in plant cells,
such as Lemna. Methods for production of antibodies in a plant system are
disclosed in the U.S. patent application corresponding to Alston & Bird
LLP attorney docket No. 040989/314911, filed on Aug. 11, 2006. PCT
Publication WO 99/54342 by Umana et al. describes cell lines engineered
to express glycoprotein-modifying glycosyl transferases (e.g.,
beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that
antibodies expressed in the engineered cell lines exhibit increased
bisecting GlcNac structures which results in increased ADCC activity of
the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).
Alternatively, the fucose residues of the antibody may be cleaved off
using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase
removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975)
Biochem. 14:5516-23).

[0235] Another modification of the antibodies herein that is contemplated
by this disclosure is pegylation. An antibody can be pegylated to, for
example, increase the biological (e.g., serum) half life of the antibody.
To pegylate an antibody, the antibody, or fragment thereof, typically is
reacted with polyethylene glycol (PEG), such as a reactive ester or
aldehyde derivative of PEG, under conditions in which one or more PEG
groups become attached to the antibody or antibody fragment. Preferably,
the pegylation is carried out via an acylation reaction or an alkylation
reaction with a reactive PEG molecule (or an analogous reactive
water-soluble polymer). As used herein, the term "polyethylene glycol" is
intended to encompass any of the forms of PEG that have been used to
derivatize other proteins, such as mono (C1-C10) alkoxy- or
aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain
embodiments, the antibody to be pegylated is an aglycosylated antibody.
Methods for pegylating proteins are known in the art and can be applied
to the antibodies of this disclosure. See for example, EP 0 154 316 by
Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Antibody Fragments and Antibody Mimetics

[0236] The instant invention is not limited to traditional antibodies and
may be practiced through the use of antibody fragments and antibody
mimetics. As detailed below, a wide variety of antibody fragment and
antibody mimetic technologies have now been developed and are widely
known in the art. While a number of these technologies, such as domain
antibodies, Nanobodies, and UniBodies make use of fragments of, or other
modifications to, traditional antibody structures, there are also
alternative technologies, such as Affibodies, DARPins, Anticalins,
Avimers, and Versabodies that employ binding structures that, while they
mimic traditional antibody binding, are generated from and function via
distinct mechanisms.

[0237] Domain Antibodies (dAbs) are the smallest functional binding units
of antibodies, corresponding to the variable regions of either the heavy
(VH) or light (VL) chains of human antibodies. Domain Antibodies have a
molecular weight of approximately 13 kDa. Domantis has developed a series
of large and highly functional libraries of fully human VH and VL dAbs
(more than ten billion different sequences in each library), and uses
these libraries to select dAbs that are specific to therapeutic targets.
In contrast to many conventional antibodies, Domain Antibodies are well
expressed in bacterial, yeast, and mammalian cell systems. Further
details of domain antibodies and methods of production thereof may be
obtained by reference to U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081;
6,172,197; 6,696,245; US Serial No. 2004/0110941; European patent
application No. 1433846 and European Patents 0368684 & 0616640;
WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 and
WO03/002609, each of which is herein incorporated by reference in its
entirety.

[0238] Nanobodies are antibody-derived therapeutic proteins that contain
the unique structural and functional properties of naturally-occurring
heavy-chain antibodies. These heavy-chain antibodies contain a single
variable domain (VHH) and two constant domains (CH2 and CH3).
Importantly, the cloned and isolated VHH domain is a perfectly stable
polypeptide harbouring the full antigen-binding capacity of the original
heavy-chain antibody. Nanobodies have a high homology with the VH domains
of human antibodies and can be further humanized without any loss of
activity. Importantly, Nanobodies have a low immunogenic potential, which
has been confirmed in primate studies with Nanobody lead compounds.

[0239] Nanobodies combine the advantages of conventional antibodies with
important features of small molecule drugs. Like conventional antibodies,
Nanobodies show high target specificity, high affinity for their target
and low inherent toxicity. However, like small molecule drugs they can
inhibit enzymes and readily access receptor clefts. Furthermore,
Nanobodies are extremely stable, can be administered by means other than
injection (see e.g. WO 04/041867, which is herein incorporated by
reference in its entirety) and are easy to manufacture. Other advantages
of Nanobodies include recognizing uncommon or hidden epitopes as a result
of their small size, binding into cavities or active sites of protein
targets with high affinity and selectivity due to their unique
3-dimensional, drug format flexibility, tailoring of half-life and ease
and speed of drug discovery.

[0240] Nanobodies are encoded by single genes and are efficiently produced
in almost all prokaryotic and eukaryotic hosts e.g. E. coli (see e.g.
U.S. Pat. No. 6,765,087, which is herein incorporated by reference in its
entirety), molds (for example Aspergillus or Trichoderma) and yeast (for
example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see e.g. U.S.
Pat. No. 6,838,254, which is herein incorporated by reference in its
entirety). The production process is scalable and multi-kilogram
quantities of Nanobodies have been produced. Because Nanobodies exhibit a
superior stability compared with conventional antibodies, they can be
formulated as a long shelf-life, ready-to-use solution.

[0241] The Nanoclone method (see e.g. WO 06/079372, which is herein
incorporated by reference in its entirety) is a proprietary method for
generating Nanobodies against a desired target, based on automated
high-throughout selection of B-cells and could be used in the context of
the instant invention.

[0242] UniBodies are another antibody fragment technology, however this
one is based upon the removal of the hinge region of IgG4 antibodies. The
deletion of the hinge region results in a molecule that is essentially
half the size of traditional IgG4 antibodies and has a univalent binding
region rather than the bivalent binding region of IgG4 antibodies. It is
also well known that IgG4 antibodies are inert and thus do not interact
with the immune system, which may be advantageous for the treatment of
diseases where an immune response is not desired, and this advantage is
passed onto UniBodies. For example, UniBodies may function to inhibit or
silence, but not kill, the cells to which they are bound. Additionally,
UniBody binding to cancer cells do not stimulate them to proliferate.
Furthermore, because UniBodies are about half the size of traditional
IgG4 antibodies, they may show better distribution over larger solid
tumors with potentially advantageous efficacy. UniBodies are cleared from
the body at a similar rate to whole IgG4 antibodies and are able to bind
with a similar affinity for their antigens as whole antibodies. Further
details of UniBodies may be obtained by reference to patent
WO2007/059782, which is herein incorporated by reference in its entirety.

[0243] Affibody molecules represent a new class of affinity proteins based
on a 58-amino acid residue protein domain, derived from one of the
IgG-binding domains of staphylococcal protein A. This three helix bundle
domain has been used as a scaffold for the construction of combinatorial
phagemid libraries, from which Affibody variants that target the desired
molecules can be selected using phage display technology (Nord K,
Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren P A, Binding
proteins selected from combinatorial libraries of an α-helical
bacterial receptor domain, Nat Biotechnol 1997; 15:772-7. Ronmark J,
Gronlund H, Uhlen M, Nygren P A, Human immunoglobulin A (IgA)-specific
ligands from combinatorial engineering of protein A, Eur J Biochem 2002;
269:2647-55.). The simple, robust structure of Affibody molecules in
combination with their low molecular weight (6 kDa), make them suitable
for a wide variety of applications, for instance, as detection reagents
(Ronmark J, Hansson M, Nguyen T, et al, Construction and characterization
of affibody-Fc chimeras produced in Escherichia coli, J Immunol Methods
2002; 261:199-211) and to inhibit receptor interactions (Sandstorm K, Xu
Z, Forsberg G, Nygren P A, Inhibition of the CD28-CD80 co-stimulation
signal by a CD28-binding Affibody ligand developed by combinatorial
protein engineering, Protein Eng 2003; 16:691-7). Further details of
Affibodies and methods of production thereof may be obtained by reference
to U.S. Pat. No. 5,831,012 which is herein incorporated by reference in
its entirety.

[0244] Labelled Affibodies may also be useful in imaging applications for
determining abundance of Isoforms.

[0245] DARPins (Designed Ankyrin Repeat Proteins) are one example of an
antibody mimetic DRP (Designed Repeat Protein) technology that has been
developed to exploit the binding abilities of non-antibody polypeptides.
Repeat proteins such as ankyrin or leucine-rich repeat proteins, are
ubiquitous binding molecules, which occur, unlike antibodies, intra- and
extracellularly. Their unique modular architecture features repeating
structural units (repeats), which stack together to form elongated repeat
domains displaying variable and modular target-binding surfaces. Based on
this modularity, combinatorial libraries of polypeptides with highly
diversified binding specificities can be generated. This strategy
includes the consensus design of self-compatible repeats displaying
variable surface residues and their random assembly into repeat domains.

[0246] DARPins can be produced in bacterial expression systems at very
high yields and they belong to the most stable proteins known. Highly
specific, high-affinity DARPins to a broad range of target proteins,
including human receptors, cytokines, kinases, human proteases, viruses
and membrane proteins, have been selected. DARPins having affinities in
the single-digit nanomolar to picomolar range can be obtained.

[0247] DARPins have been used in a wide range of applications, including
ELISA, sandwich ELISA, flow cytometric analysis (FACS),
immunohistochemistry (IHC), chip applications, affinity purification or
Western blotting. DARPins also proved to be highly active in the
intracellular compartment for example as intracellular marker proteins
fused to green fluorescent protein (GFP). DARPins were further used to
inhibit viral entry with IC50 in the pM range. DARPins are not only ideal
to block protein-protein interactions, but also to inhibit enzymes.
Proteases, kinases and transporters have been successfully inhibited,
most often an allosteric inhibition mode. Very fast and specific
enrichments on the tumor and very favorable tumor to blood ratios make
DARPins well suited for in vivo diagnostics or therapeutic approaches.

[0248] Additional information regarding DARPins and other DRP technologies
can be found in US Patent Application Publication No. 2004/0132028 and
International Patent Application Publication No. WO 02/20565, both of
which are hereby incorporated by reference in their entirety.

[0249] Anticalins are an additional antibody mimetic technology, however
in this case the binding specificity is derived from lipocalins, a family
of low molecular weight proteins that are naturally and abundantly
expressed in human tissues and body fluids. Lipocalins have evolved to
perform a range of functions in vivo associated with the physiological
transport and storage of chemically sensitive or insoluble compounds.
Lipocalins have a robust intrinsic structure comprising a highly
conserved β-barrel which supports four loops at one terminus of the
protein. These loops form the entrance to a binding pocket and
conformational differences in this part of the molecule account for the
variation in binding specificity between individual lipocalins.

[0250] While the overall structure of hypervariable loops supported by a
conserved β-sheet framework is reminiscent of immunoglobulins,
lipocalins differ considerably from antibodies in terms of size, being
composed of a single polypeptide chain of 160-180 amino acids which is
marginally larger than a single immunoglobulin domain.

[0251] Lipocalins are cloned and their loops are subjected to engineering
in order to create Anticalins. Libraries of structurally diverse
Anticalins have been generated and Anticalin display allows the selection
and screening of binding function, followed by the expression and
production of soluble protein for further analysis in prokaryotic or
eukaryotic systems. Studies have successfully demonstrated that
Anticalins can be developed that are specific for virtually any human
target protein can be isolated and binding affinities in the nanomolar or
higher range can be obtained.

[0252] Anticalins can also be formatted as dual targeting proteins,
so-called Duocalins. A Duocalin binds two separate therapeutic targets in
one easily produced monomeric protein using standard manufacturing
processes while retaining target specificity and affinity regardless of
the structural orientation of its two binding domains.

[0253] Modulation of multiple targets through a single molecule is
particularly advantageous in diseases known to involve more than a single
causative factor. Moreover, bi- or multivalent binding formats such as
Duocalins have significant potential in targeting cell surface molecules
in disease, mediating agonistic effects on signal transduction pathways
or inducing enhanced internalization effects via binding and clustering
of cell surface receptors. Furthermore, the high intrinsic stability of
Duocalins is comparable to monomeric Anticalins, offering flexible
formulation and delivery potential for Duocalins.

[0254] Additional information regarding Anticalins can be found in U.S.
Pat. No. 7,250,297 and International Patent Application Publication No.
WO 99/16873, both of which are hereby incorporated by reference in their
entirety.

[0255] Another antibody mimetic technology useful in the context of the
instant invention are Avimers. Avimers are evolved from a large family of
human extracellular receptor domains by in vitro exon shuffling and phage
display, generating multidomain proteins with binding and inhibitory
properties. Linking multiple independent binding domains has been shown
to create avidity and results in improved affinity and specificity
compared with conventional single-epitope binding proteins. Other
potential advantages include simple and efficient production of
multitarget-specific molecules in Escherichia coli, improved
thermostability and resistance to proteases. Avimers with sub-nanomolar
affinities have been obtained against a variety of targets.

[0256] Additional information regarding Avimers can be found in US Patent
Application Publication Nos. 2006/0286603, 2006/0234299, 2006/0223114,
2006/0177831, 2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932,
2005/0053973, 2005/0048512, 2004/0175756, all of which are hereby
incorporated by reference in their entirety.

[0257] Versabodies are another antibody mimetic technology that could be
used in the context of the instant invention. Versabodies are small
proteins of 3-5 kDa with >15% cysteines, which form a high disulfide
density scaffold, replacing the hydrophobic core that typical proteins
have. The replacement of a large number of hydrophobic amino acids,
comprising the hydrophobic core, with a small number of disulfides
results in a protein that is smaller, more hydrophilic (less aggregation
and non-specific binding), more resistant to proteases and heat, and has
a lower density of T-cell epitopes, because the residues that contribute
most to MHC presentation are hydrophobic. All four of these properties
are well-known to affect immunogenicity, and together they are expected
to cause a large decrease in immunogenicity.

[0258] The inspiration for Versabodies comes from the natural injectable
biopharmaceuticals produced by leeches, snakes, spiders, scorpions,
snails, and anemones, which are known to exhibit unexpectedly low
immunogenicity. Starting with selected natural protein families, by
design and by screening the size, hydrophobicity, proteolytic antigen
processing, and epitope density are minimized to levels far below the
average for natural injectable proteins.

[0259] Given the structure of Versabodies, these antibody mimetics offer a
versatile format that includes multi-valency, multi-specificity, a
diversity of half-life mechanisms, tissue targeting modules and the
absence of the antibody Fc region. Furthermore, Versabodies are
manufactured in E. coli at high yields, and because of their
hydrophilicity and small size, Versabodies are highly soluble and can be
formulated to high concentrations. Versabodies are exceptionally heat
stable (they can be boiled) and offer extended shelf-life.

[0260] Additional information regarding Versabodies can be found in US
Patent Application Publication No. 2007/0191272 which is hereby
incorporated by reference in its entirety.

[0261] The detailed description of antibody fragment and antibody mimetic
technologies provided above is not intended to be a comprehensive list of
all technologies that could be used in the context of the instant
specification. For example, and also not by way of limitation, a variety
of additional technologies including alternative polypeptide-based
technologies, such as fusions of complimentary determining regions as
outlined in Qui et al., Nature Biotechnology, 25(8) 921-929 (2007), which
is hereby incorporated by reference in its entirety, as well as nucleic
acid-based technologies, such as the RNA aptamer technologies described
in U.S. Pat. Nos. 5,789,157, 5,864,026, 5,712,375, 5,763,566, 6,013,443,
6,376,474, 6,613,526, 6,114,120, 6,261,774, and 6,387,620, all of which
are hereby incorporated by reference, could be used in the context of the
instant invention.

Antibody Physical Properties

[0262] The antibodies of the present disclosure may be further
characterized by the various physical properties of the anti-CXCR4
antibodies. Various assays may be used to detect and/or differentiate
different classes of antibodies based on these physical properties.

[0263] In some embodiments, antibodies of the present disclosure may
contain one or more glycosylation sites in either the light or heavy
chain variable region. The presence of one or more glycosylation sites in
the variable region may result in increased immunogenicity of the
antibody or an alteration of the pK of the antibody due to altered
antigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala
F A and Morrison S L (2004) J Immunol 172:5489-94; Wallick et al (1988) J
Exp Med 168:1099-109; Spiro R G (2002) Glycobiology 12:43R-56R; Parekh et
al (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).
Glycosylation has been known to occur at motifs containing an N-X-S/T
sequence. Variable region glycosylation may be tested using a Glycoblot
assay, which cleaves the antibody to produce a Fab, and then tests for
glycosylation using an assay that measures periodate oxidation and Schiff
base formation. Alternatively, variable region glycosylation may be
tested using Dionex light chromatography (Dionex-LC), which cleaves
saccharides from a Fab into monosaccharides and analyzes the individual
saccharide content. In some instances, it is preferred to have an
anti-CXCR4 antibody that does not contain variable region glycosylation.
This can be achieved either by selecting antibodies that do not contain
the glycosylation motif in the variable region or by mutating residues
within the glycosylation motif using standard techniques well known in
the art.

[0264] In a preferred embodiment, the antibodies of the present disclosure
do not contain asparagine isomerism sites. A deamidation or isoaspartic
acid effect may occur on N-G or D-G sequences, respectively. The
deamidation or isoaspartic acid effect results in the creation of
isoaspartic acid which decreases the stability of an antibody by creating
a kinked structure off a side chain carboxy terminus rather than the main
chain.

[0265] The creation of isoaspartic acid can be measured using an iso-quant
assay, which uses a reverse-phase HPLC to test for isoaspartic acid.

[0266] Each antibody will have a unique isoelectric point (pI), but
generally antibodies will fall in the pH range of between 6 and 9.5. The
pI for an IgG1 antibody typically falls within the pH range of 7-9.5 and
the pI for an IgG4 antibody typically falls within the pH range of 6-8.
Antibodies may have a pI that is outside this range. Although the effects
are generally unknown, there is speculation that antibodies with a pI
outside the normal range may have some unfolding and instability under in
vivo conditions. The isoelectric point may be tested using a capillary
isoelectric focusing assay, which creates a pH gradient and may utilize
laser focusing for increased accuracy (Janini et al (2002)
Electrophoresis 23:1605-11; Ma et al. (2001) Chromatographia 53:S75-89;
Hunt et al (1998) J Chromatogr A 800:355-67). In some instances, it is
preferred to have an anti-CXCR4 antibody that contains a pI value that
falls in the normal range. This can be achieved either by selecting
antibodies with a pI in the normal range, or by mutating charged surface
residues using standard techniques well known in the art.

[0267] Each antibody will have a melting temperature that is indicative of
thermal stability (Krishnamurthy R and Manning M C (2002) Curr Pharm
Biotechnol 3:361-71). A higher thermal stability indicates greater
overall antibody stability in vivo. The melting point of an antibody may
be measure using techniques such as differential scanning calorimetry
(Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol
Lett 68:47-52). TM1 indicates the temperature of the initial
unfolding of the antibody. TM2 indicates the temperature of complete
unfolding of the antibody. Generally, it is preferred that the TM1
of an antibody of the present disclosure is greater than 60° C.,
preferably greater than 65° C., even more preferably greater than
70° C. Alternatively, the thermal stability of an antibody may be
measure using circular dichroism (Murray et al. (2002) J. Chromatogr Sci
40:343-9).

[0268] In a preferred embodiment, antibodies are selected that do not
rapidly degrade. Fragmentation of an anti-CXCR4 antibody may be measured
using capillary electrophoresis (CE) and MALDI-MS, as is well understood
in the art (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

[0269] In another preferred embodiment, antibodies are selected that have
minimal aggregation effects. Aggregation may lead to triggering of an
unwanted immune response and/or altered or unfavorable pharmacokinetic
properties. Generally, antibodies are acceptable with aggregation of 25%
or less, preferably 20% or less, even more preferably 15% or less, even
more preferably 10% or less and even more preferably 5% or less.
Aggregation may be measured by several techniques well known in the art,
including size-exclusion column (SEC) high performance liquid
chromatography (HPLC), and light scattering to identify monomers, dimers,
trimers or multimers.

Methods of Engineering Antibodies

[0270] As discussed above, the anti-CXCR4 antibodies having VH and
VK sequences disclosed herein can be used to create new anti-CXCR4
antibodies by modifying the VH and/or VK sequences, or the
constant region(s) attached thereto. Thus, in another aspect of this
disclosure, the structural features of an anti-CXCR4 antibody of this
disclosure, e.g. F7, F9, D1 or E2, are used to create structurally
related anti-CXCR4 antibodies that retain at least one functional
property of the antibodies of this disclosure, such as binding to human
CXCR4. For example, one or more CDR regions of F7, F9, D1 or E2, or
mutations thereof, can be combined recombinantly with known framework
regions and/or other CDRs to create additional, recombinantly-engineered,
anti-CXCR4 antibodies of this disclosure, as discussed above. Other types
of modifications include those described in the previous section. The
starting material for the engineering method is one or more of the
VH and/or VK sequences provided herein, or one or more CDR
regions thereof. To create the engineered antibody, it is not necessary
to actually prepare (i.e., express as a protein) an antibody having one
or more of the VH and/or VK sequences provided herein, or one
or more CDR regions thereof. Rather, the information contained in the
sequence(s) is used as the starting material to create a "second
generation" sequence(s) derived from the original sequence(s) and then
the "second generation" sequence(s) is prepared and expressed as a
protein.

[0271] Accordingly, in another embodiment, this disclosure provides a
method for preparing an anti-CXCR4 antibody comprising:

[0272] (a)
providing: (i) a heavy chain variable region antibody sequence comprising
a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-4, a
CDR2 sequence selected from the group consisting of SEQ ID NOs: 5-8,
and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs:
9-12; and/or (ii) a light chain variable region antibody sequence
comprising a CDR1 sequence selected from the group consisting of SEQ ID
NOs: 13-16, a CDR2 sequence selected from the group consisting of SEQ ID
NOs: 17-20, and/or a CDR3 sequence selected from the group consisting of
SEQ ID NOs: 21-24;

[0275] Standard molecular biology techniques can be used to prepare and
express the altered antibody sequence.

[0276] Preferably, the antibody encoded by the altered antibody
sequence(s) is one that retains one, some or all of the functional
properties of the anti-CXCR4 antibodies described herein, which
functional properties include, but are not limited to:

[0287] (xi)
increasing survival time of a CXCR4.sup.+ tumor-bearing subject.

[0288] The functional properties of the altered antibodies can be assessed
using standard assays available in the art and/or described herein, such
as those set forth in the Examples (e.g., flow cytometry, binding assays,
functional assays).

[0289] In certain embodiments of the methods of engineering antibodies of
this disclosure, mutations can be introduced randomly or selectively
along all or part of an anti-CXCR4 antibody coding sequence and the
resulting modified anti-CXCR4 antibodies can be screened for binding
activity and/or other functional properties as described herein.
Mutational methods have been described in the art. For example, PCT
Publication WO 02/092780 by Short describes methods for creating and
screening antibody mutations using saturation mutagenesis, synthetic
ligation assembly, or a combination thereof. Alternatively, PCT
Publication WO 03/074679 by Lazar et al. describes methods of using
computational screening methods to optimize physiochemical properties of
antibodies.

Nucleic Acid Molecules Encoding Antibodies of this Disclosure

[0290] Another aspect of this disclosure pertains to nucleic acid
molecules that encode the antibodies of this disclosure. The nucleic
acids may be present in whole cells, in a cell lysate, or in a partially
purified or substantially pure form. A nucleic acid is "isolated" or
"rendered substantially pure" when purified away from other cellular
components or other contaminants, e.g., other cellular nucleic acids or
proteins, by standard techniques, including alkaline/SDS treatment, CsCl
banding, column chromatography, agarose gel electrophoresis and others
well known in the art. See, F. Ausubel, et al., ed. (1987) Current
Protocols in Molecular Biology, Greene Publishing and Wiley Interscience,
New York. A nucleic acid of this disclosure can be, for example, DNA or
RNA and may or may not contain intronic sequences. In a preferred
embodiment, the nucleic acid is a cDNA molecule.

[0291] Nucleic acids of this disclosure can be obtained using standard
molecular biology techniques. For antibodies expressed by hybridomas
(e.g., hybridomas prepared from transgenic mice carrying human
immunoglobulin genes as described further below), cDNAs encoding the
light and heavy chains of the antibody made by the hybridoma can be
obtained by standard PCR amplification or cDNA cloning techniques. For
antibodies obtained from an immunoglobulin gene library (e.g., using
phage display techniques), a nucleic acid encoding such antibodies can be
recovered from the gene library.

[0293] Once DNA fragments encoding VH and VL segments are
obtained, these DNA fragments can be further manipulated by standard
recombinant DNA techniques, for example to convert the variable region
genes to full-length antibody chain genes, to Fab fragment genes or to a
scFv gene. In these manipulations, a VL- or VH-encoding DNA
fragment is operatively linked to another DNA fragment encoding another
protein, such as an antibody constant region or a flexible linker. The
term "operatively linked", as used in this context, is intended to mean
that the two DNA fragments are joined such that the amino acid sequences
encoded by the two DNA fragments remain in-frame.

[0294] The isolated DNA encoding the VH region can be converted to a
full-length heavy chain gene by operatively linking the VH-encoding DNA
to another DNA molecule encoding heavy chain constant regions (CH1, CH2
and CH3). The sequences of human heavy chain constant region genes are
known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department of
Health and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR amplification.
The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA,
IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG4
constant region. For a Fab fragment heavy chain gene, the
VH-encoding DNA can be operatively linked to another DNA molecule
encoding only the heavy chain CH1 constant region.

[0295] The isolated DNA encoding the VL region can be converted to a
full-length light chain gene (as well as a Fab light chain gene) by
operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of human
light chain constant region genes are known in the art (see e.g., Kabat,
E. A., et al. (1991) Sequences of Proteins of Immunological Interest,
Fifth Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242) and DNA fragments encompassing these regions can
be obtained by standard PCR amplification. In preferred embodimients, the
light chain constant region can be a kappa or lambda constant region.

[0297] Monoclonal antibodies (mAbs) of the present disclosure can be
produced by a variety of techniques, including conventional monoclonal
antibody methodology e.g., the standard somatic cell hybridization
technique of Kohler and Milstein (1975) Nature 256: 495. Although somatic
cell hybridization procedures are preferred, in principle, other
techniques for producing monoclonal antibody can be employed e.g., viral
or oncogenic transformation of B lymphocytes.

[0298] The preferred animal system for preparing hybridomas is the murine
system. Hybridoma production in the mouse is a very well-established
procedure. Immunization protocols and techniques for isolation of
immunized splenocytes for fusion are known in the art. Fusion partners
(e.g., murine myeloma cells) and fusion procedures are also known.

[0299] Chimeric or humanized antibodies of the present disclosure can be
prepared based on the sequence of a non-human monoclonal antibody
prepared as described above. DNA encoding the heavy and light chain
immunoglobulins can be obtained from the non-human hybridoma of interest
and engineered to contain non-murine (e.g., human) immunoglobulin
sequences using standard molecular biology techniques. For example, to
create a chimeric antibody, murine variable regions can be linked to
human constant regions using methods known in the art (see e.g., U.S.
Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,
murine CDR regions can be inserted into a human framework using methods
known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.
Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0300] In a preferred embodiment, the antibodies of this disclosure are
human monoclonal antibodies. Such human monoclonal antibodies directed
against CXCR4 can be generated using transgenic or transchromosomic mice
carrying parts of the human immune system rather than the mouse system.
These transgenic and transchromosomic mice include mice referred to
herein as the HuMAb Mouse® and KM Mouse®, respectively, and are
collectively referred to herein as "human Ig mice."

[0302] In another embodiment, human antibodies of this disclosure can be
raised using a mouse that carries human immunoglobulin sequences on
transgenes and transchomosomes, such as a mouse that carries a human
heavy chain transgene and a human light chain transchromosome. This mouse
is referred to herein as a "KM Mouse®" and is described in detail in
PCT Publication WO 02/43478 to Ishida et al.

[0303] Still further, alternative transgenic animal systems expressing
human immunoglobulin genes are available in the art and can be used to
raise anti-CXCR4 antibodies of this disclosure. For example, an
alternative transgenic system referred to as the Xenomouse (Abgenix,
Inc.) can be used; such mice are described in, for example, U.S. Pat.
Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 to
Kucherlapati et al.

[0304] Moreover, alternative transchromosomic animal systems expressing
human immunoglobulin genes are available in the art and can be used to
raise anti-CXCR4 antibodies of this disclosure. For example, mice
carrying both a human heavy chain transchromosome and a human light chain
tranchromosome, referred to as "TC mice" can be used; such mice are
described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA
97:722-727. Furthermore, cows carrying human heavy and light chain
transchromosomes have been described in the art (e.g., Kuroiwa et al.
(2002) Nature Biotechnology 20:889-894 and PCT application No. WO
2002/092812) and can be used to raise anti-CXCR4 antibodies of this
disclosure.

[0305] Human monoclonal antibodies of this disclosure can also be prepared
using phage display methods for screening libraries of human
immunoglobulin genes. Such phage display methods for isolating human
antibodies are established in the art. See for example: U.S. Pat. Nos.
5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos.
5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and
6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;
6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

[0306] Human monoclonal antibodies of this disclosure can also be prepared
using SCID mice into which human immune cells have been reconstituted
such that a human antibody response can be generated upon immunization.
Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and
5,698,767 to Wilson et al.

[0307] In a particularly preferred embodiment, human anti-CXCR4 antibodies
are prepared using a combination of human Ig mouse and phage display
techniques, as described in U.S. Pat. No. 6,794,132 by Buechler et al.
More specifically, the method first involves raising an anti-CXCR4
antibody response in a human Ig mouse (such as a HuMab mouse or KM mouse
as described above) by immunizing the mouse with a CXCR4 antigen,
followed by isolating nucleic acids encoding human antibody chains from
lymphatic cells of the mouse and introducing these nucleic acids into a
display vector (e.g., phage) to provide a library of display packages.
Thus, each library member comprises a nucleic acid encoding a human
antibody chain and each antibody chain is displayed from the display
package. The library then is screened with a CXCR4 antigen to isolate
library members that specifically bind CXCR4. Nucleic acid inserts of the
selected library members then are isolated and sequenced by standard
methods to determine the light and heavy chain variable sequences of the
selected CXCR4 binders. The variable regions can be converted to
full-length antibody chains by standard recombinant DNA techniques, such
as cloning of the variable regions into an expression vector that carries
the human heavy and light chain constant regions such that the VH region
is operatively linked to the CH region and the VL region is operatively
linked to the CL region. For a further description of the preparation of
human anti-CXCR4 antibodies using this combined transgenic mouse/phage
display system, see Example 1.

Immunization of Human Ig Mice

[0308] When human Ig mice are used to raise human antibodies of this
disclosure, such mice can be immunized with a purified or enriched
preparation of CXCR4 antigen and/or recombinant CXCR4, or cells
expressing CXCR4, or a CXCR4 fusion protein, as described by Lonberg, N.
et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996)
Nature Biotechnology 14: 845-851; and PCT Publication WO 98/24884 and WO
01/14424. Preferably, the mice will be 6-16 weeks of age upon the first
infusion. For example, a purified or recombinant preparation (5-50 μg)
of CXCR4 antigen can be used to immunize the human Ig mice
intraperitoneally. Most preferably, the immunogen used to raise the
antibodies of this disclosure comprises human CXCR4 in its native
conformation within a membrane, non-limiting examples of which include
cells transfected to express CXCR4 on their cell surface, cells that
natively express CXCR4 (e.g., CEM cells), and artificial membranes (e.g.,
liposomes) into which CXCR4 has been incorporated, such as magnetic
proteoliposomes (MPLs) that incorporate CXCR4 (described further in
Example 1).

[0309] Detailed procedures to generate fully human monoclonal antibodies
to CXCR4 are described in Example 1 below. Cumulative experience with
various antigens has shown that the transgenic mice respond when
initially immunized intraperitoneally (IP) with antigen in complete
Freund's adjuvant, followed by every other week IP immunizations (up to a
total of 6) with antigen in incomplete Freund's adjuvant. However,
adjuvants other than Freund's are also found to be effective. In
addition, whole cells in the absence of adjuvant are found to be highly
immunogenic. The immune response can be monitored over the course of the
immunization protocol with plasma samples being obtained by retroorbital
bleeds. The plasma can be screened by ELISA (as described below), and
mice with sufficient titers of anti-CXCR4 human immunoglobulin can be
used for fusions. Mice can be boosted intravenously with antigen 3 days
before sacrifice and removal of the spleen. It is expected that 2-3
fusions for each immunization may need to be performed. Between 6 and 24
mice are typically immunized for each antigen. Usually both HCo7 and
HCo12 strains are used. In addition, both HCo7 and HCo12 transgene can be
bred together into a single mouse having two different human heavy chain
transgenes (HCo7/HCo12). Alternatively or additionally, the KM Mouse®
strain can be used, as described in Example 1.

Generation of Hybridomas Producing Human Monoclonal Antibodies of this
Disclosure

[0310] To generate hybridomas producing human monoclonal antibodies of
this disclosure, splenocytes and/or lymph node cells from immunized mice
can be isolated and fused to an appropriate immortalized cell line, such
as a mouse myeloma cell line. The resulting hybridomas can be screened
for the production of antigen-specific antibodies. For example, single
cell suspensions of splenic lymphocytes from immunized mice can be fused
to one-sixth the number of P3×63-Ag8.653 nonsecreting mouse myeloma
cells (ATCC, CRL 1580) with 50% PEG. Alternatively, the single cell
suspension of splenic lymphocytes from immunized mice can be fused using
an electric field based electrofusion method, using a CytoPulse large
chamber cell fusion electroporator (CytoPulse Sciences, Inc., Glen Burnie
Md.). Cells are plated at approximately 2×105 in flat bottom
microtiter plate, followed by a two week incubation in selective medium
containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen
(IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM
2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50
mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after
the fusion). After approximately two weeks, cells can be cultured in
medium in which the HAT is replaced with HT. Individual wells can then be
screened by ELISA for human monoclonal IgM and IgG antibodies. Once
extensive hybridoma growth occurs, medium can be observed usually after
10-14 days. The antibody secreting hybridomas can be replated, screened
again, and if still positive for human IgG, the monoclonal antibodies can
be subcloned at least twice by limiting dilution. The stable subclones
can then be cultured in vitro to generate small amounts of antibody in
tissue culture medium for characterization.

[0311] To purify human monoclonal antibodies, selected hybridomas can be
grown in two-liter spinner-flasks for monoclonal antibody purification.
Supernatants can be filtered and concentrated before affinity
chromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).
Eluted IgG can be checked by gel electrophoresis and high performance
liquid chromatography to ensure purity. The buffer solution can be
exchanged into PBS, and the concentration can be determined by OD280
using 1.43 extinction coefficient. The monoclonal antibodies can be
aliquoted and stored at -80° C.

Generation of Transfectomas Producing Monoclonal Antibodies of this
Disclosure

[0312] Antibodies of this disclosure also can be produced in a host cell
transfectoma using, for example, a combination of recombinant DNA
techniques and gene transfection methods as is well known in the art
(e.g., Morrison, S. (1985) Science 229:1202).

[0313] For example, to express the antibodies, or antibody fragments
thereof, DNAs encoding partial or full-length light and heavy chains, can
be obtained by standard molecular biology techniques (e.g., PCR
amplification or cDNA cloning using a hybridoma that expresses the
antibody of interest) and the DNAs can be inserted into expression
vectors such that the genes are operatively linked to transcriptional and
translational control sequences. In this context, the term "operatively
linked" is intended to mean that an antibody gene is ligated into a
vector such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression vector
and expression control sequences are chosen to be compatible with the
expression host cell used. The antibody light chain gene and the antibody
heavy chain gene can be inserted into separate vector or, more typically,
both genes are inserted into the same expression vector. The antibody
genes are inserted into the expression vector by standard methods (e.g.,
ligation of complementary restriction sites on the antibody gene fragment
and vector, or blunt end ligation if no restriction sites are present).
The light and heavy chain variable regions of the antibodies described
herein can be used to create full-length antibody genes of any antibody
isotype by inserting them into expression vectors already encoding heavy
chain constant and light chain constant regions of the desired isotype
such that the VH segment is operatively linked to the CH
segment(s) within the vector and the VK segment is operatively
linked to the CL segment within the vector. Additionally or
alternatively, the recombinant expression vector can encode a signal
peptide that facilitates secretion of the antibody chain from a host
cell. The antibody chain gene can be cloned into the vector such that the
signal peptide is linked in-frame to the amino terminus of the antibody
chain gene. The signal peptide can be an immunoglobulin signal peptide or
a heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).

[0314] In addition to the antibody chain genes, the recombinant expression
vectors of this disclosure carry regulatory sequences that control the
expression of the antibody chain genes in a host cell. The term
"regulatory sequence" is intended to include promoters, enhancers and
other expression control elements (e.g., polyadenylation signals) that
control the transcription or translation of the antibody chain genes.
Such regulatory sequences are described, for example, in Goeddel (Gene
Expression Technology. Methods in Enzymology 185, Academic Press, San
Diego, Calif. (1990)). It will be appreciated by those skilled in the art
that the design of the expression vector, including the selection of
regulatory sequences, may depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein desired,
etc. Preferred regulatory sequences for mammalian host cell expression
include viral elements that direct high levels of protein expression in
mammalian cells, such as promoters and/or enhancers derived from
cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the
adenovirus major late promoter (AdMLP) and polyoma. Alternatively,
nonviral regulatory sequences may be used, such as the ubiquitin promoter
or β-globin promoter. Still further, regulatory elements composed of
sequences from different sources, such as the SRα promoter system,
which contains sequences from the SV40 early promoter and the long
terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al.
(1988) Mol. Cell. Biol. 8:466-472).

[0315] In addition to the antibody chain genes and regulatory sequences,
the recombinant expression vectors of this disclosure may carry
additional sequences, such as sequences that regulate replication of the
vector in host cells (e.g., origins of replication) and selectable marker
genes. The selectable marker gene facilitates selection of host cells
into which the vector has been introduced (see, e.g., U.S. Pat. Nos.
4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,
typically the selectable marker gene confers resistance to drugs, such as
G418, hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with
methotrexate selection/amplification) and the neo gene (for G418
selection). For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a host
cell by standard techniques. The various forms of the term "transfection"
are intended to encompass a wide variety of techniques commonly used for
the introduction of exogenous DNA into a prokaryotic or eukaryotic host
cell, e.g., electroporation, calcium-phosphate precipitation,
DEAE-dextran transfection and the like. Although it is theoretically
possible to express the antibodies of this disclosure in either
prokaryotic or eukaryotic host cells, expression of antibodies in
eukaryotic cells, and most preferably mammalian host cells, is the most
preferred because such eukaryotic cells, and in particular mammalian
cells, are more likely than prokaryotic cells to assemble and secrete a
properly folded and immunologically active antibody. Prokaryotic
expression of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood, C. R.
(1985) Immunology Today 6:12-13).

[0316] Preferred mammalian host cells for expressing the recombinant
antibodies of this disclosure include Chinese Hamster Ovary (CHO cells)
(including dhfr.sup.- CHO cells, described in Urlaub and Chasin, (1980)
Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable
marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J.
Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In
particular, for use with NSO myeloma cells, another preferred expression
system is the GS gene expression system disclosed in WO 87/04462 (to
Wilson), WO 89/01036 (to Bebbington) and EP 338,841 (to Bebbington). When
recombinant expression vectors encoding antibody genes are introduced
into mammalian host cells, the antibodies are produced by culturing the
host cells for a period of time sufficient to allow for expression of the
antibody in the host cells or, more preferably, secretion of the antibody
into the culture medium in which the host cells are grown. Antibodies can
be recovered from the culture medium using standard protein purification
methods.

Characterization of Antibody Binding to Antigen

[0317] Antibodies of this disclosure can be tested for binding to CXCR4
by, for example, standard flow cytometry methods. Since the antibodies of
this disclosure preferably recognize CXCR4 in its native conformation
within a membrane, testing for binding to CXCR4 preferably is done with
an assay (e.g., flow cytometry) that utilizes a reagent expressing native
conformation CXCR4. Nonlimiting examples of reagents expressing native
conformation CXCR4 that can be used in the binding assays include cells
that naturally express CXCR4 (e.g., CEM cells), cells that have been
transfected to express CXCR4 (e.g., R1610 cells transfected with a CXCR4
expression vector) and liposomes into which CXCR4 has been incorporated
(e.g., magnetic proteoliposomes incorporating CXCR4), each of which is
described in further detail in the Examples. Briefly, for the flow
cytometry assay, cells expressing CXCR4 are incubated with the test
antibody, washed, incubated with a labeled secondary reagent capable of
binding to the test antibody, washed again, and subjected to analysis to
detect the binding of the secondary reagent to the cells (e.g., using a
FACS machine). Preferably, mice that develop the highest titers as
evaluated by flow cytometry will be used for fusions or for further
selection of antibodies (e.g., by phage display screening of antibody
libraries made from B cells of the mouse).

[0318] A flow cytometry assay as described above can also be used to
screen for hybridomas that show positive reactivity with CXCR4 immunogen.
Hybridomas expressing antibodies that bind with high avidity to CXCR4 are
subcloned and further characterized. One clone from each hybridoma, which
retains the reactivity of the parent cells (by flow cytometry), can be
chosen for making a 5-10 vial cell bank stored at -140° C., and
for antibody purification.

[0319] To purify anti-CXCR4 antibodies, selected hybridomas can be grown
in two-liter spinner-flasks for monoclonal antibody purification.
Supernatants can be filtered and concentrated before affinity
chromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).
Eluted IgG can be checked by gel electrophoresis and high performance
liquid chromatography to ensure purity. The buffer solution can be
exchanged into PBS, and the concentration can be determined by OD280
using 1.43 extinction coefficient. The monoclonal antibodies can be
aliquoted and stored at -80° C.

[0320] To determine if the selected anti-CXCR4 monoclonal antibodies bind
to unique epitopes, each antibody can be biotinylated using commercially
available reagents (Pierce, Rockford, Ill.). Competition studies using
unlabeled monoclonal antibodies and biotinylated monoclonal antibodies
can be performed using a whole cell ELISA assay in which ELISA plates are
coated with cells expressing CXCR4, and the ability of the unlabeled
antibody to compete with the biotinylated antibody for binding to the
CXCR4-expressing cells is examined. Biotinylated mAb binding can be
detected with a strep-avidin-alkaline phosphatase probe.

[0321] To determine the isotype of purified antibodies, isotype ELISAs can
be performed using reagents specific for antibodies of a particular
isotype. For example, to determine the isotype of a human monoclonal
antibody, wells of microtiter plates can be coated with 1 μg/ml of
anti-human immunoglobulin overnight at 4° C. After blocking with
1% BSA, the plates are reacted with 1 μg/ml or less of test monoclonal
antibodies or purified isotype controls, at ambient temperature for one
to two hours. The wells can then be reacted with either human IgG1 or
human IgM-specific alkaline phosphatase-conjugated probes. Plates are
developed and analyzed as described above.

[0322] Anti-CXCR4 human IgGs can be further tested for reactivity with
CXCR4 antigen by Western blotting. Briefly, CXCR4 can be prepared and
subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis.
After electrophoresis, the separated antigens are transferred to
nitrocellulose membranes, blocked with 10% fetal calf serum, and probed
with the monoclonal antibodies to be tested. Human IgG binding can be
detected using anti-human IgG alkaline phosphatase and developed with
BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

[0323] The binding specificity of an antibody of this disclosure may also
be determined by monitoring binding of the antibody to cells expressing
CXCR4, for example by flow cytometry. Typically, a cell line, such as a
CHO cell line, may be transfected with an expression vector encoding a
transmembrane form of CXCR4. The transfected protein may comprise a tag,
such as a myc-tag, preferably at the N-terminus, for detection using an
antibody to the tag. Binding of an antibody of this disclosure to CXCR4
may be determined by incubating the transfected cells with the antibody,
and detecting bound antibody. Binding of an antibody to the tag on the
transfected protein may be used as a positive control.

[0325] Other preferred examples of therapeutic cytotoxins that can be
conjugated to an antibody of this disclosure include duocarmycins,
calicheamicins, maytansines and auristatins, and derivatives thereof. An
example of a calicheamicin antibody conjugate is commercially available
(Mylotarg®; American Home Products).

[0326] Cytotoxins can be conjugated to antibodies of this disclosure using
linker technology available in the art. Examples of linker types that
have been used to conjugate a cytotoxin to an antibody include, but are
not limited to, hydrazones, thioethers, esters, disulfides and
peptide-containing linkers. A linker can be chosen that is, for example,
susceptible to cleavage by low pH within the lysosomal compartment or
susceptible to cleavage by proteases, such as proteases preferentially
expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

[0328] Antibodies of the present disclosure also can be conjugated to a
radioactive isotope to generate cytotoxic radiopharmaceuticals, also
referred to as radioimmunoconjugates. Examples of radioactive isotopes
that can be conjugated to antibodies for use diagnostically or
therapeutically include, but are not limited to, iodine131,
indium111, yttrium90 and lutetium177. Method for preparing
radioimmunconjugates are established in the art. Examples of
radioimmunoconjugates are commercially available, including Zevalin®
(IDEC Pharmaceuticals) and Bexxar® (Corixa Pharmaceuticals), and
similar methods can be used to prepare radioimmunoconjugates using the
antibodies of this disclosure.

[0329] The antibody conjugates of this disclosure can be used to modify a
given biological response, and the drug moiety is not to be construed as
limited to classical chemical therapeutic agents. For example, the drug
moiety may be a protein or polypeptide possessing a desired biological
activity. Such proteins may include, for example, an enzymatically active
toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or
interferon-γ; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other
growth factors.

[0331] In another aspect, the present disclosure features bispecific
molecules comprising an anti-CXCR4 antibody, or a fragment thereof, of
this disclosure. An antibody of this disclosure, or antigen-binding
portions thereof, can be derivatized or linked to another functional
molecule, e.g., another peptide or protein (e.g., another antibody or
ligand for a receptor) to generate a bispecific molecule that binds to at
least two different binding sites or target molecules. The antibody of
this disclosure may in fact be derivatized or linked to more than one
other functional molecule to generate multispecific molecules that bind
to more than two different binding sites and/or target molecules; such
multispecific molecules are also intended to be encompassed by the term
"bispecific molecule" as used herein. To create a bispecific molecule of
this disclosure, an antibody of this disclosure can be functionally
linked (e.g., by chemical coupling, genetic fusion, noncovalent
association or otherwise) to one or more other binding molecules, such as
another antibody, antibody fragment, peptide or binding mimetic, such
that a bispecific molecule results.

[0332] Accordingly, the present disclosure includes bispecific molecules
comprising at least one first binding specificity for CXCR4 and a second
binding specificity for a second target epitope. In a particular
embodiment of this disclosure, the second target epitope is an Fc
receptor, e.g., human FcγRI (CD64) or a human Fcα receptor
(CD89). Therefore, this disclosure includes bispecific molecules capable
of binding both to FcγR or FcαR expressing effector cells
(e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to
target cells expressing CXCR4. These bispecific molecules target CXCR4
expressing cells to effector cell and trigger Fc receptor-mediated
effector cell activities, such as phagocytosis of CXCR4 expressing cells,
antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release,
or generation of superoxide anion.

[0333] In an embodiment of this disclosure in which the bispecific
molecule is multispecific, the molecule can further include a third
binding specificity, in addition to an anti-Fc binding specificity and an
anti-CXCR4 binding specificity. In one embodiment, the third binding
specificity is an anti-enhancement factor (EF) portion, e.g., a molecule
which binds to a surface protein involved in cytotoxic activity and
thereby increases the immune response against the target cell. The
"anti-enhancement factor portion" can be an antibody, functional antibody
fragment or a ligand that binds to a given molecule, e.g., an antigen or
a receptor, and thereby results in an enhancement of the effect of the
binding determinants for the Fc receptor or target cell antigen. The
"anti-enhancement factor portion" can bind an Fc receptor or a target
cell antigen. Alternatively, the anti-enhancement factor portion can bind
to an entity that is different from the entity to which the first and
second binding specificities bind. For example, the anti-enhancement
factor portion can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28,
CD4, CD40, ICAM-1 or other immune cell that results in an increased
immune response against the target cell).

[0334] In one embodiment, the bispecific molecules of this disclosure
comprise as a binding specificity at least one antibody, or an antibody
fragment thereof, including, e.g., an Fab, Fab', F(ab')2, Fv, Fd,
dAb or a single chain Fv. The antibody may also be a light chain or heavy
chain dimer, or any minimal fragment thereof such as a Fv or a single
chain construct as described in U.S. Pat. No. 4,946,778 to Ladner et al.,
the contents of which is expressly incorporated by reference.

[0335] In one embodiment, the binding specificity for an Fcγ
receptor is provided by a monoclonal antibody, the binding of which is
not blocked by human immunoglobulin G (IgG). As used herein, the term
"IgG receptor" refers to any of the eight γ-chain genes located on
chromosome 1. These genes encode a total of twelve transmembrane or
soluble receptor isoforms which are grouped into three Fcγ receptor
classes: FcγRI (CD64), FcγRII (CD32), and FcγRIII
(CD16). In one preferred embodiment, the Fcγ receptor a human high
affinity FcγRI. The human FcγRI is a 72 kDa molecule, which
shows high affinity for monomeric IgG (108-109 M-1).

[0336] The production and characterization of certain preferred
anti-Fcγ, monoclonal antibodies are described in PCT Publication WO
88/00052 and in U.S. Pat. No. 4,954,617 to Fanger et al., the teachings
of which are fully incorporated by reference herein. These antibodies
bind to an epitope of FcγRI, FcγRII or FcγRIII at a
site which is distinct from the Fcγ binding site of the receptor
and, thus, their binding is not blocked substantially by physiological
levels of IgG. Specific anti-FcγRI antibodies useful in this
disclosure are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma
producing mAb 32 is available from the American Type Culture Collection,
ATCC Accession No. HB9469. In other embodiments, the anti-Fcγ
receptor antibody is a humanized form of monoclonal antibody 22 (H22).
The production and characterization of the H22 antibody is described in
Graziano, R. F. et al. (1995) J. Immunol. 155 (10): 4996-5002 and PCT
Publication WO 94/10332 to Tempest et al. The H22 antibody producing cell
line was deposited at the American Type Culture Collection under the
designation HA022CL1 and has the accession no. CRL 11177.

[0337] In still other preferred embodiments, the binding specificity for
an Fc receptor is provided by an antibody that binds to a human IgA
receptor, e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of
which is preferably not blocked by human immunoglobulin A (IgA). The term
"IgA receptor" is intended to include the gene product of one
α-gene (FcαRI) located on chromosome 19. This gene is known
to encode several alternatively spliced transmembrane isoforms of 55 to
110 kDa. FcαRI (CD89) is constitutively expressed on
monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but
not on non-effector cell populations. FcαRI has medium affinity
(≈5×107 M-1) for both IgA1 and IgA2, which is
increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H.
C. et al. (1996) Critical Reviews in Immunology 16:423-440). Four
FcαRI-specific monoclonal antibodies, identified as A3, A59, A62
and A77, which bind FcαRI outside the IgA ligand binding domain,
have been described (Monteiro, R. C. et al. (1992) J. Immunol. 148:1764).

[0338] FcαRI and FcγRI are preferred trigger receptors for use
in the bispecific molecules of this disclosure because they are (1)
expressed primarily on immune effector cells, e.g., monocytes, PMNs,
macrophages and dendritic cells; (2) expressed at high levels (e.g.,
5,000-100,000 per cell); (3) mediators of cytotoxic activities (e.g.,
ADCC, phagocytosis); and (4) mediate enhanced antigen presentation of
antigens, including self-antigens, targeted to them.

[0339] While human monoclonal antibodies are preferred, other antibodies
which can be employed in the bispecific molecules of this disclosure are
murine, chimeric and humanized monoclonal antibodies.

[0340] The bispecific molecules of the present disclosure can be prepared
by conjugating the constituent binding specificities, e.g., the anti-FcR
and anti-CXCR4 binding specificities, using methods known in the art. For
example, each binding specificity of the bispecific molecule can be
generated separately and then conjugated to one another. When the binding
specificities are proteins or peptides, a variety of coupling or
cross-linking agents can be used for covalent conjugation. Examples of
cross-linking agents include protein A, carbodiimide,
N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic
acid) (DTNB), o-phenylenedimaleimide (oPDM),
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;
Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods
include those described in Paulus (1985) Behring Ins. Mitt. No. 78,
118-132; Brennan et al. (1985) Science 229:81-83, and Glennie et al.
(1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA
and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

[0341] When the binding specificities are antibodies, they can be
conjugated via sulfhydryl bonding of the C-terminus hinge regions of the
two heavy chains. In a particularly preferred embodiment, the hinge
region is modified to contain an odd number of sulfhydryl residues,
preferably one, prior to conjugation.

[0342] Alternatively, both binding specificities can be encoded in the
same vector and expressed and assembled in the same host cell. This
method is particularly useful where the bispecific molecule is a
mAb×mAb, mAb×Fab, Fab×F(ab')2 or ligand x Fab
fusion protein. A bispecific molecule of this disclosure can be a single
chain molecule comprising one single chain antibody and a binding
determinant, or a single chain bispecific molecule comprising two binding
determinants. Bispecific molecules may comprise at least two single chain
molecules. Methods for preparing bispecific molecules are described for
example in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405;
5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858, all of which
are expressly incorporated herein by reference.

[0343] Binding of the bispecific molecules to their specific targets can
be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth
inhibition), or Western Blot assay. Each of these assays generally
detects the presence of protein-antibody complexes of particular interest
by employing a labeled reagent (e.g., an antibody) specific for the
complex of interest. For example, the FcR-antibody complexes can be
detected using e.g., an enzyme-linked antibody or antibody fragment which
recognizes and specifically binds to the antibody-FcR complexes.
Alternatively, the complexes can be detected using any of a variety of
other immunoassays. For example, the antibody can be radioactively
labeled and used in a radioimmunoassay (RIA) (see, for example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course
on Radioligand Assay Techniques, The Endocrine Society, March, 1986,
which is incorporated by reference herein). The radioactive isotope can
be detected by such means as the use of a γ counter or a
scintillation counter or by autoradiography.

Pharmaceutical Compositions

[0344] In another aspect, the present disclosure provides a composition,
e.g., a pharmaceutical composition, containing one or a combination of
monoclonal antibodies, or antigen-binding portion(s) thereof, of the
present disclosure, formulated together with a pharmaceutically
acceptable carrier. Such compositions may include one or a combination of
(e.g., two or more different) antibodies, or immunoconjugates or
bispecific molecules of this disclosure. For example, a pharmaceutical
composition of this disclosure can comprise a combination of antibodies
(or immunoconjugates or bispecifics) that bind to different epitopes on
the target antigen or that have complementary activities.

[0345] Pharmaceutical compositions of this disclosure also can be
administered in combination therapy, i.e., combined with other agents.
For example, the combination therapy can include an anti-CXCR4 antibody
of the present disclosure combined with at least one other
anti-inflammatory or immunosuppressant agent. Examples of therapeutic
agents that can be used in combination therapy are described in greater
detail below in the section on uses of the antibodies of this disclosure.

[0346] As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the like
that are physiologically compatible. Preferably, the carrier is suitable
for intravenous, intramuscular, subcutaneous, parenteral, spinal or
epidermal administration (e.g., by injection or infusion). Depending on
the route of administration, the active compound, i.e., antibody,
immunoconjugate, or bispecific molecule, may be coated in a material to
protect the compound from the action of acids and other natural
conditions that may inactivate the compound.

[0347] The pharmaceutical compounds of this disclosure may include one or
more pharmaceutically acceptable salts. A "pharmaceutically acceptable
salt" refers to a salt that retains the desired biological activity of
the parent compound and does not impart any undesired toxicological
effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19).
Examples of such salts include acid addition salts and base addition
salts. Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,
hydroiodic, phosphorous and the like, as well as from nontoxic organic
acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted
alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include those
derived from alkaline earth metals, such as sodium, potassium, magnesium,
calcium and the like, as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, procaine and the like.

[0349] Examples of suitable aqueous and nonaqueous carriers that may be
employed in the pharmaceutical compositions of this disclosure include
water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable oils,
such as olive oil, and injectable organic esters, such as ethyl oleate.
Proper fluidity can be maintained, for example, by the use of coating
materials, such as lecithin, by the maintenance of the required particle
size in the case of dispersions, and by the use of surfactants.

[0350] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing agents.
Prevention of presence of microorganisms may be ensured both by
sterilization procedures, supra, and by the inclusion of various
antibacterial and antifungal agents, for example, paraben, chlorobutanol,
phenol sorbic acid, and the like. It may also be desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into the
compositions. In addition, prolonged absorption of the injectable
pharmaceutical form may be brought about by the inclusion of agents which
delay absorption such as aluminum monostearate and gelatin.

[0351] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use of
such media and agents for pharmaceutically active substances is known in
the art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the pharmaceutical
compositions of this disclosure is contemplated. Supplementary active
compounds can also be incorporated into the compositions.

[0352] Therapeutic compositions typically must be sterile and stable under
the conditions of manufacture and storage. The composition can be
formulated as a solution, microemulsion, liposome, or other ordered
structure suitable to high drug concentration. The carrier can be a
solvent or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper fluidity
can be maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars, polyalcohols
such as mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about
by including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.

[0353] Sterile injectable solutions can be prepared by incorporating the
active compound in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required, followed
by sterilization microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that contains a
basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation are
vacuum drying and freeze-drying (lyophilization) that yield a powder of
the active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.

[0354] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary depending upon
the subject being treated, and the particular mode of administration. The
amount of active ingredient which can be combined with a carrier material
to produce a single dosage form will generally be that amount of the
composition which produces a therapeutic effect. Generally, out of one
hundred percent, this amount will range from about 0.01 percent to about
ninety-nine percent of active ingredient, preferably from about 0.1
percent to about 70 percent, most preferably from about 1 percent to
about 30 percent of active ingredient in combination with a
pharmaceutically acceptable carrier.

[0355] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single bolus may
be administered, several divided doses may be administered over time or
the dose may be proportionally reduced or increased as indicated by the
exigencies of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used herein
refers to physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity of
active compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The specification
for the dosage unit forms of this disclosure are dictated by and directly
dependent on (a) the unique characteristics of the active compound and
the particular therapeutic effect to be achieved, and (b) the limitations
inherent in the art of compounding such an active compound for the
treatment of sensitivity in individuals.

[0356] For administration of the antibody, the dosage ranges from about
0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body
weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body
weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight
or within the range of 1-10 mg/kg. An exemplary treatment regime entails
administration once per week, once every two weeks, once every three
weeks, once every four weeks, once a month, once every 3 months or once
every three to 6 months. Preferred dosage regimens for an anti-CXCR4
antibody of this disclosure include 1 mg/kg body weight or 3 mg/kg body
weight via intravenous administration, with the antibody being given
using one of the following dosing schedules: (i) every four weeks for six
dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg
body weight once followed by 1 mg/kg body weight every three weeks.

[0357] In some methods, two or more monoclonal antibodies with different
binding specificities are administered simultaneously, in which case the
dosage of each antibody administered falls within the ranges indicated.
Antibody is usually administered on multiple occasions. Intervals between
single dosages can be, for example, weekly, monthly, every three monthgs
or yearly. Intervals can also be irregular as indicated by measuring
blood levels of antibody to the target antigen in the patient. In some
methods, dosage is adjusted to achieve a plasma antibody concentration of
about 1-1000 μg/ml and in some methods about 25-300 μg/ml.

[0358] Alternatively, antibody can be administered as a sustained release
formulation, in which case less frequent administration is required.
Dosage and frequency vary depending on the half-life of the antibody in
the patient. In general, human antibodies show the longest half life,
followed by humanized antibodies, chimeric antibodies, and nonhuman
antibodies. The dosage and frequency of administration can vary depending
on whether the treatment is prophylactic or therapeutic. In prophylactic
applications, a relatively low dosage is administered at relatively
infrequent intervals over a long period of time. Some patients continue
to receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short intervals is
sometimes required until progression of the disease is reduced or
terminated, and preferably until the patient shows partial or complete
amelioration of symptoms of disease. Thereafter, the patient can be
administered a prophylactic regime.

[0359] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present disclosure may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient. The selected dosage level will depend upon a variety of
pharmacokinetic factors including the activity of the particular
compositions of the present disclosure employed, or the ester, salt or
amide thereof, the route of administration, the time of administration,
the rate of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials used
in combination with the particular compositions employed, the age, sex,
weight, condition, general health and prior medical history of the
patient being treated, and like factors well known in the medical arts.

[0360] A "therapeutically effective dosage" of an anti-CXCR4 antibody of
this disclosure preferably results in a decrease in severity of disease
symptoms, an increase in frequency and duration of disease symptom-free
periods, or a prevention of impairment or disability due to the disease
affliction. For example, for the treatment of CXCR4.sup.+ tumors, a
"therapeutically effective dosage" preferably inhibits cell growth or
tumor growth by at least about 20%, more preferably by at least about
40%, even more preferably by at least about 60%, and still more
preferably by at least about 80% relative to untreated subjects. The
ability of a compound to inhibit tumor growth can be evaluated in an
animal model system predictive of efficacy in human tumors.
Alternatively, this property of a composition can be evaluated by
examining the ability of the compound to inhibit cell growth, such
inhibition can be measured in vitro by assays known to the skilled
practitioner. A therapeutically effective amount of a therapeutic
compound can decrease tumor size, or otherwise ameliorate symptoms in a
subject. One of ordinary skill in the art would be able to determine such
amounts based on such factors as the subject's size, the severity of the
subject's symptoms, and the particular composition or route of
administration selected.

[0361] A composition of the present disclosure can be administered via one
or more routes of administration using one or more of a variety of
methods known in the art. As will be appreciated by the skilled artisan,
the route and/or mode of administration will vary depending upon the
desired results. Preferred routes of administration for antibodies of
this disclosure include intravenous, intramuscular, intradermal,
intraperitoneal, subcutaneous, spinal or other parenteral routes of
administration, for example by injection or infusion. The phrase
"parenteral administration" as used herein means modes of administration
other than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular, intraarterial,
intrathecal, intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural and
intrasternal injection and infusion.

[0362] Alternatively, an antibody of this disclosure can be administered
via a non-parenteral route, such as a topical, epidermal or mucosal route
of administration, for example, intranasally, orally, vaginally,
rectally, sublingually or topically.

[0363] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible polymers
can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally known to
those skilled in the art. See, e.g., Sustained and Controlled Release
Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New
York, 1978.

[0364] Therapeutic compositions can be administered with medical devices
known in the art. For example, in a preferred embodiment, a therapeutic
composition of this disclosure can be administered with a needleless
hypodermic injection device, such as the devices disclosed in U.S. Pat.
Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or
4,596,556. Examples of well-known implants and modules useful in the
present disclosure include: U.S. Pat. No. 4,487,603, which discloses an
implantable micro-infusion pump for dispensing medication at a controlled
rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat. No.
4,475,196, which discloses an osmotic drug delivery system. These patents
are incorporated herein by reference. Many other such implants, delivery
systems, and modules are known to those skilled in the art.

[0366] The antibodies, particulary the human antibodies, antibody
compositions and methods of the present disclosure have numerous in vitro
and in vivo diagnostic and therapeutic utilities involving the diagnosis
and treatment of CXCR4 mediated disorders. For example, these molecules
can be administered to cells in culture, in vitro or ex vivo, or to human
subjects, e.g., in vivo, to treat, prevent and to diagnose a variety of
disorders. As used herein, the term "subject" is intended to include
human and non-human animals. Non-human animals include all vertebrates,
e.g., mammals and non-mammals, such as non-human primates, sheep, dogs,
cats, cows, horses, chickens, amphibians, and reptiles. Preferred
subjects include human patients having disorders mediated by or modulated
by CXCR4 activity or involving the CXCR4/SDF-1 pathway. When antibodies
to CXCR4 are administered together with another agent, the two can be
administered in either order or simultaneously.

[0367] Given the specific binding of the antibodies of this disclosure for
CXCR4, the antibodies of this disclosure can be used to specifically
detect CXCR4 expression on the surface of cells and, moreover, can be
used to purify CXCR4 via immunoaffinity purification.

[0368] Suitable routes of administering the antibody compositions (e.g.,
human monoclonal antibodies, multispecific and bispecific molecules and
immunoconjugates) of this disclosure in vivo and in vitro are well known
in the art and can be selected by those of ordinary skill. For example,
the antibody compositions can be administered by injection (e.g.,
intravenous or subcutaneous). Suitable dosages of the molecules used will
depend on the age and weight of the subject and the concentration and/or
formulation of the antibody composition.

[0369] As previously described, human anti-CXCR4 antibodies of this
disclosure can be co-administered with one or other more therapeutic
agents, e.g., a cytotoxic agent, a radiotoxic agent or an
immunosuppressive agent. The antibody can be linked to the agent (as an
immunocomplex) or can be administered separate from the agent. In the
latter case (separate administration), the antibody can be administered
before, after or concurrently with the agent or can be co-administered
with other known therapies, e.g., an anti-cancer therapy, e.g.,
radiation. Such therapeutic agents include, among others, anti-neoplastic
agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate,
carmustine, chlorambucil, and cyclophosphamide hydroxyurea which, by
themselves, are only effective at levels which are toxic or subtoxic to a
patient. Cisplatin is intravenously administered as a 100 mg/kg dose once
every four weeks and adriamycin is intravenously administered as a 60-75
mg/ml dose once every 21 days. Co-administration of the human anti-CXCR4
antibodies, or antigen binding fragments thereof, of the present
disclosure with chemotherapeutic agents provides two anti-cancer agents
which operate via different mechanisms which yield a cytotoxic effect to
human tumor cells. Such co-administration can solve problems due to
development of resistance to drugs or a change in the antigenicity of the
tumor cells that would render them unreactive with the antibody.

[0370] Target-specific effector cells, e.g., effector cells linked to
compositions (e.g., human antibodies, multispecific and bispecific
molecules) of this disclosure can also be used as therapeutic agents.
Effector cells for targeting can be human leukocytes such as macrophages,
neutrophils or monocytes. Other cells include eosinophils, natural killer
cells and other IgG- or IgA-receptor bearing cells. If desired, effector
cells can be obtained from the subject to be treated. The target-specific
effector cells can be administered as a suspension of cells in a
physiologically acceptable solution. The number of cells administered can
be in the order of 108-109 but will vary depending on the
therapeutic purpose. In general, the amount will be sufficient to obtain
localization at the target cell, e.g., a tumor cell expressing CXCR4, and
to effect cell killing by, e.g., phagocytosis. Routes of administration
can also vary.

[0371] Therapy with target-specific effector cells can be performed in
conjunction with other techniques for removal of targeted cells. For
example, anti-tumor therapy using the compositions (e.g., human
antibodies, multispecific and bispecific molecules) of this disclosure
and/or effector cells armed with these compositions can be used in
conjunction with chemotherapy. Additionally, combination immunotherapy
may be used to direct two distinct cytotoxic effector populations toward
tumor cell rejection. For example, anti-CXCR4 antibodies linked to
anti-Fc-gamma R1 or anti-CD3 may be used in conjunction with IgG- or
IgA-receptor specific binding agents.

[0372] Bispecific and multispecific molecules of this disclosure can also
be used to modulate FcγR or FcγR levels on effector cells,
such as by capping and elimination of receptors on the cell surface.
Mixtures of anti-Fc receptors can also be used for this purpose.

[0373] The compositions (e.g., human, humanized, or chimeric antibodies,
multispecific and bispecific molecules and immunoconjugates) of this
disclosure which have complement binding sites, such as portions from
IgG1, -2, or -3 or IgM which bind complement, can also be used in the
presence of complement. In one embodiment, ex vivo treatment of a
population of cells comprising target cells with a binding agent of this
disclosure and appropriate effector cells can be supplemented by the
addition of complement or serum containing complement. Phagocytosis of
target cells coated with a binding agent of this disclosure can be
improved by binding of complement proteins. In another embodiment target
cells coated with the compositions (e.g., human antibodies, multispecific
and bispecific molecules) of this disclosure can also be lysed by
complement. In yet another embodiment, the compositions of this
disclosure do not activate complement.

[0374] The compositions (e.g., human, humanized, or chimeric antibodies,
multispecific and bispecific molecules and immunoconjugates) of this
disclosure can also be administered together with complement.
Accordingly, within the scope of this disclosure are compositions
comprising human antibodies, multispecific or bispecific molecules and
serum or complement. These compositions are advantageous in that the
complement is located in close proximity to the human antibodies,
multispecific or bispecific molecules. Alternatively, the human
antibodies, multispecific or bispecific molecules of this disclosure and
the complement or serum can be administered separately.

[0375] The antibodies of this disclosure also can be used in combination
with one or more additional therapeutic antibodies or other binding
agents, such as Ig fusion proteins. Non-limiting examples of other
antibodies or binding agents with which an anti-CXCR4 antibody of this
disclosure can be administered in combination include antibodies or
binding agents to CTLA-4, PSMA, CD30, IP-10, IFN-γ, CD70, PD-1,
PD-L1, TNF, TNF-R, VEGF, VEGF-R, CCR5, IL-1, IL-18, IL-18R, CD19,
Campath-1, EGFR, CD33, CD20, Her-2, CD25, gpIIb/IIIa, IgE, CD11a,
α4 integrin, IFNα and IFNAR1.

[0376] Also within the scope of the present disclosure are kits comprising
the antibody compositions of this disclosure (e.g., human antibodies,
bispecific or multispecific molecules, or immunoconjugates) and
instructions for use. The kit can further contain one ore more additional
reagents, such as an immunosuppressive reagent, a cytotoxic agent or a
radiotoxic agent, or one or more additional human antibodies of this
disclosure (e.g., a human antibody having a complementary activity which
binds to an epitope in the CXCR4 antigen distinct from the first human
antibody).

[0377] Accordingly, patients treated with antibody compositions of this
disclosure can be additionally administered (prior to, simultaneously
with, or following administration of a human antibody of this disclosure)
with another therapeutic agent, such as a cytotoxic or radiotoxic agent,
which enhances or augments the therapeutic effect of the human
antibodies.

[0378] In other embodiments, the subject can be additionally treated with
an agent that modulates, e.g., enhances or inhibits, the expression or
activity of Fcγ or Fcγ receptors by, for example, treating
the subject with a cytokine. Preferred cytokines for administration
during treatment with the multispecific molecule include of granulocyte
colony-stimulating factor (G-CSF), granulocyte-macrophage
colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and
tumor necrosis factor (TNF).

[0379] The compositions (e.g., human antibodies, multispecific and
bispecific molecules) of this disclosure can also be used to target cells
expressing CXCR4, for example for labeling such cells. For such use, the
binding agent can be linked to a molecule that can be detected. Thus,
this disclosure provides methods for localizing ex vivo or in vitro cells
expressing CXCR4. The detectable label can be, e.g., a radioisotope, a
fluorescent compound, an enzyme, or an enzyme co-factor.

[0380] In a particular embodiment, this disclosure provides methods for
detecting the presence of CXCR4 antigen in a sample, or measuring the
amount of CXCR4 antigen, comprising contacting the sample, and a control
sample, with a human monoclonal antibody, or an antigen binding portion
thereof, which specifically binds to CXCR4, under conditions that allow
for formation of a complex between the antibody or portion thereof and
CXCR4. The formation of a complex is then detected, wherein a difference
complex formation between the sample compared to the control sample is
indicative the presence of CXCR4 antigen in the sample.

[0381] In yet another embodiment, immunoconjugates of this disclosure can
be used to target compounds (e.g., therapeutic agents, labels,
cytotoxins, radiotoxoins immunosuppressants, etc.) to cells which have
CXCR4 cell surface receptors by linking such compounds to the antibody.
Thus, this disclosure also provides methods for localizing ex vivo or in
vivo cells expressing CXCR4 (e.g., with a detectable label, such as a
radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor).
Alternatively, the immunoconjugates can be used to kill cells which have
CXCR4 cell surface receptors by targeting cytotoxins or radiotoxins to
CXCR4.

[0382] CXCR4 is known to be expressed on a wide variety of tumor cells
types and also is known to be involved in tumor metastasis. Moreover, as
a coreceptor for HIV entry into T cells, CXCR4 is known to be involved in
HIV infection. Additionally, the CXCR4/SDF-1 pathway has been shown to be
involved in inflammatory conditions. Still further, the CXCR4/SDF-1
pathway has been shown to be involved in angiogenesis or
neovascularization. Accordingly, the anti-CXCR4 antibodies (and
immunoconjugates and bispecific molecules) of this disclosure can be used
to modulate CXCR4 activity in each of these clinical situations, as
follows:

[0385] In view of the foregoing, the anti-CXCR4 antibodies of this
disclosure can be used in the treatment of cancers, including but not
limited to a cancer selected from the group consisting of breast,
ovarian, prostate, non-small cell lung, pancreatic, thyroid,
nasopharyngeal carcinoma, melanoma, renal cell carcinoma, lymphoma,
neuroblastoma, glioblastoma, rhabdomyosarcoma, colorectal, kidney,
osteosarcoma, acute lymphoblastic leukemia and acute myeloid leukemia.
The antibody can be used alone or in combination other cancer treatments,
such as surgery and/or radiation, and/or with other anti-neoplastic
agents, such as the anti-neoplastic agents discussed and set forth above,
including chemotherapeutic drugs and other anti-tumor antigen antibodies,
such as those that bind CD20, Her2, PSMA, Campath-1, EGFR and the like.

[0386] B. Viral Infections, Including HIV Infection

[0387] CXCR4 has been shown to be a coreceptor for HIV entry into T cells
and, additionally, certain murine anti-CXCR4 antibodies have been
demonstrated to be able to inhibit entry of HIV isolates into T cells
(see Hou, T. et al. (1998) J. Immunol. 160:180-188; Carnec, X. et al.
(2005) J. Virol. 79:1930-1938). Thus, CXCR4 can be used as a receptor by
viruses for entry into the cell and antibodies to CXCR4 can be used to
inhibit cell entry of such viruses that use CXCR4 as a receptor.
Accordingly, the human anti-CXCR4 antibodies of this disclosure can be
used to inhibit entry of a virus into a cell, wherein the virus uses
CXCR4 as a receptor for cell entry, such that viral infection is
inhibited. In a preferred embodiment, the antibodies are used to inhibit
entry of HIV into T cells, e.g., in the treatment or prevention of
HIV/AIDS. The antibody can be used alone or in combination with other
anti-viral agents, such as anti-retroviral drugs such as AZT or protease
inhibitors.

[0392] It has been demonstrated that SDF-1 induces neovascularization
through recruitment of CXCR4-expressing hemangiocytes (Jin, D. K. et al.
(2006) Nat. Med. 12:557-567). Moreover, blockade of the SDF-1/CXCR4
pathway can attenuate in vivo tumor growth by inhibiting angiogenesis in
a VEGF-independent manner (Guleng, B. et al. (2005) Cancer Res.
65:5864-58-71). Still further, as demonstrated in Example 2, antibodies
of this disclosure are capable of inhibiting capillary tube formation in
vitro. Accordingly, the anti-CXCR4 antibodies of this disclosure that
inhibit binding of SDF-1 to CXCR4 can be used to inhibit angiogenesis by
interfering with the SDF-1/CXCR4 pathway Inhibition of angiogenesis can
be used, for example, to inhibit tumor growth or tumor metastasis
(regardless of whether the tumor is CXCR4.sup.+). The antibody can be
used alone or in combination with other anti-angiogenic agents, such as
anti-VEGF antibodies.

[0393] E. Autologous Stem Cell Transplantation

[0394] Peripheral blood stem cells are the preferred source of stem cells
for use in autologous stem cell transplantion, for example in the
treatment of certain hematological malignancies. Collection of stem cells
from the peripheral blood requires mobilization of CD34.sup.+ stem cells
from the bone marrow to the peripheral blood. Various cytokines,
chemokines and adhesion molecules have been implicated in the regulation
of this process (reviewed in Gazitt, Y. (2001) J. Hematother. Stem Cell
Res. 10:229-236), including the interaction of CXCR4 and SDF-1. Moreover,
a small molecule CXCR4 antagonist has been demonstrated to stimulate
rapid mobilization of CD34.sup.+ stem cells from the bone marrow to the
periphery (see e.g., Devine, S. M. et al. (2004) J. Clin. Oncol.
22:1095-1102; Broxmeyer, H. E. et al. (2005) J. Exp. Med. 201:1307-1318;
Flomenberg, N. et al. (2005) Blood 106:1867-1874). Accordingly,
anti-CXCR4 antibodies of this disclosure that inhibit CXCR4 activity
(i.e., antagonist antibodies) can be used to stimulate mobilization of
CD34.sup.+ stem cells from the bone marrow to the peripheral blood to
allow for the use of such stem cells in transplantation (e.g., autologous
transplantation), for example in the treatment of hematological
disorders, such as multiple myeloma and non-Hodgkin's lymphoma. The
antibody can be used alone or in combination with other agents used to
stimulate mobilization of stem cells, such as G-CSF and/or GM-CSF. Thus,
in another embodiment, the invention provides a method of stimulating
mobilization of CD34.sup.+ stem cells from bone marrow to peripheral
blood in a subject, the method comprising administering to the subject an
anti-CXCR4 antibody of the invention such that mobilization of CD34.sup.+
stem cells from bone marrow to peripheral blood is stimulated. The method
can further comprise collecting CD34+ stem cells from peripheral blood,
such as for use in autologous stem cell transplantation.

[0395] The present disclosure is further illustrated by the following
examples, which should not be construed as further limiting. The contents
of all figures and all references, patents and published patent
applications cited throughout this application are expressly incorporated
herein by reference.

EXAMPLES

Example 1

Generation of Human Monoclonal Antibodies Against CXCR4

[0396] Anti-CXCR4 human monoclonal antibodies were generated using a
combination approach in which, first, mice expressing human antibody
genes were immunized to raise in the mice a repertoire of human
immunoglobulins specific for human CXCR4 and then, second, a human
antibody library was prepared from spleen cells of the mice and displayed
on phage such that the phage were then screened for expression of
antibodies with specificity for CXCR4. This combination approach is
generally described in U.S. Application No. 20030091995 by Buechler et
al.

[0397] Antigen

[0398] R1610 cells (a Chinese Hamster lung cell line, originally described
in Thirion, J. P. et al. (1976) Genetics 83:137-147) were transfected
with an expression vector encoding the full-length human CXCR4 protein
such that the protein was expressed on the surface of the cells. A
codon-optimized form of the CXCR4 cDNA was used in the expression vector,
which was prepared as described in Mirzabekov, T. et al. (1999) J. Biol.
Chem. 274:28745-28750. To enhance the immunogenicity of the cells, the
cells were coated with trinitrophenol (TNP), by incubation with an
aqueous solution of trinitrobenzenesulfonic acid (TNBS), available
commercially as a 5% solution (Sigma, Cat. #P2297). More specifically,
1×108 cells were washed once with sterile PBS, incubated with
50 μl of the commercial 5% TNBS solution for one hour in the dark at
room temperature and then washed three times with PBS. The resultant
TNP-coated, CXCR-4-expressing R1610 cells were used as antigen for
immunization. The final immunogen was a mix of 100 μl of TNP-coated,
washed cells (1×107 cells) plus 100 μl of Ribi adjuvant.
Mice received six doses of the immunogen over time.

[0399] Transgenic Transchromosomic KM Mouse® Strain

[0400] Fully human monoclonal antibodies to CXCR4 were prepared by
initially immunizing the KM strain of transgenic transchromosomic mice,
which expresses human antibody genes. In this mouse strain, the
endogenous mouse kappa light chain gene has been homozygously disrupted
as described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenous
mouse heavy chain gene has been homozygously disrupted as described in
Example 1 of PCT Publication WO 01/09187. Additoinally, this mouse strain
carries a human kappa light chain transgene, KCo5 (as described in
Fishwild et al. (1996) Nature Biotechnology 14:845-851) and also contains
the SC20 transchromosome, which carries the human Ig heavy chain locus,
as described in PCT Publication WO 02/43478. KM mice are also described
in detail in U.S. Application No. 20020199213.

[0401] KM Immunization

[0402] To raise fully human monoclonal antibodies to CXCR4, mice of the KM
Mouse® strain were immunized with R1610 cells transfected to express
CXCR4 and coated with TNP (as described above for the antigen). General
immunization schemes for the raising human antibodies in mice strains
carrying human antibody genes are described in Lonberg, N. et al (1994)
Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature
Biotechnology 14: 845-851 and PCT Publication WO 98/24884. The mice were
6-16 weeks of age upon the first infusion of antigen.

[0403] KM mice were immunized with antigen in Ribi adjuvant either
intraperitonealy (IP), subcutaneously (Sc) or via footpad (FP), followed
by 3-21 days IP, Sc or FP reimmunization (for a total of 6 immunizations)
with the antigen in Ribi adjuvant. The immune response was monitored by
retroorbital bleeds. The plasma was screened by FACS staining of
CXCR4-expressing R1610 cells (versus untransfected parental R1610 cells).
Mice with sufficient titers of anti-CXCR4 human immunogolobulin were used
for harvesting spleens.

[0404] Preparation of Phage Display Library and Screening for
Anti-CXCR4Antibodies

[0405] Spleens harvested from the immunized mice described above were used
to make a phage display library expressing human antibody heavy and light
chains. More specifically total RNA was isolated from the spleens, cDNA
was prepared from the RNA and human antibody variable region cDNA was
specifically amplified by PCR, essentially as described in U.S. Patent
Application 20030091995 by Buechler et al. The library of human antibody
variable regions was cloned into phage expression vectors, again
essentially as described in U.S. Patent Application 20030091995 by
Buechler et al. The phage display library was screened for library
members having affinity for CXCR4 by panning with human CXCR4
incorporated into magnetic proteoliposomes (CXCR4-MPL). MPLs expressing
CXCR4, or other seven transmembrane (7TM) receptors (e.g., CCR5), such
that the native conformation of the 7TM receptor is maintained, have been
described previously (see e.g., Mirzabekov, T. et al. (2000) Nat.
Biotechnol. 18:649-654; Babcock, G. J. et al. (2001) J. Biol. Chem.
276:38433-38440; PCT Publication WO 01/49265; U.S. Patent Application
20010034432). In brief, recombinant human CXCR4 that contained an epitope
tag was solublized from a transfected CXCR4-expressing cell line using
the detergent CHAPSO and the protein was captured on magentic beads via
the epitope tag. A lipid membrane was reconstituted during removal of the
detergent, such that the native membrane conformation of CXCR4 was
maintained, to create the CXCR4-MPLs. Three rounds of panning of the
phage display library on the CXCR4-MPLs led to a 30-fold enrichment of
CXCR4-binders as compared to background. Variable region fragments of
interest were recloned into a Fab expression vector and the Fab retested
for antigen binding against transfected CXCR4-expressing cells. Whole
antibodies were then generated from the Fabs using standard molecular
biology techniques.

[0406] Fab clones F7, F9, D1 and E2 were selected for further analysis.

[0407] The cDNA sequences encoding the heavy and light chain variable
regions of the F7, F9, D1 and E2 Fab clones, obtained from phage display
library screening as described in Example 1, were sequenced using
standard DNA sequencing techniques.

[0408] The nucleotide and amino acid sequences of the heavy chain variable
region of F7 are shown in FIG. 1A and in SEQ ID NO: 33 and 25,
respectively.

[0409] The nucleotide and amino acid sequences of the light chain variable
region of F7 are shown in FIG. 1B and in SEQ ID NO: 37 and 29,
respectively.

[0410] Comparison of the F7 heavy chain immunoglobulin sequence to the
known human germline immunoglobulin heavy chain sequences demonstrated
that the F7 heavy chain utilizes a VH segment from human germline
VH 3-48, a D segment from the human germline 4-23, and a JH segment
from human germline JH 6B. Further analysis of the F7 VH sequence
using the Kabat system of CDR region determination led to the delineation
of the heavy chain CDR1, CDR2 and CD3 regions as shown in FIG. 1A and in
SEQ ID NOs: 1, 5 and 9, respectively.

[0411] Comparison of the F7 light chain immunoglobulin sequence to the
known human germline immunoglobulin light chain sequences demonstrated
that the F7 light chain utilizes a VL segment from human germline
VK L15 and a JK segment from human germline JK 1. Further analysis
of the F7 VL sequence using the Kabat system of CDR region
determination led to the delineation of the light chain CDR1, CDR2 and
CD3 regions as shown in FIG. 1B and in SEQ ID NOs: 13, 17 and 21,
respectively.

[0412] The nucleotide and amino acid sequences of the heavy chain variable
region of F9 are shown in FIG. 2A and in SEQ ID NO: 34 and 26,
respectively.

[0413] The nucleotide and amino acid sequences of the light chain variable
region of F9 are shown in FIG. 2B and in SEQ ID NO: 38 and 30,
respectively.

[0414] Comparison of the F9 heavy chain immunoglobulin sequence to the
known human germline immunoglobulin heavy chain sequences demonstrated
that the F9 heavy chain utilizes a VH segment from human germline
VH 3-48, a D segment from the human germline 4-23, and a JH segment
from human germline JH 6B. Further analysis of the F9 VH sequence
using the Kabat system of CDR region determination led to the delineation
of the heavy chain CDR1, CDR2 and CD3 regions as shown in FIG. 2A and in
SEQ ID NOs: 2, 6 and 10, respectively.

[0415] Comparison of the F9 light chain immunoglobulin sequence to the
known human germline immunoglobulin light chain sequences demonstrated
that the F9 light chain utilizes a VL segment from human germline
VK L15 and a JK segment from human germline JK 1. Further analysis
of the F9 VL sequence using the Kabat system of CDR region
determination led to the delineation of the light chain CDR1, CDR2 and
CD3 regions as shown in FIG. 2B and in SEQ ID NOs: 14, 18 and 22,
respectively.

[0416] The nucleotide and amino acid sequences of the heavy chain variable
region of D1 are shown in FIG. 3A and in SEQ ID NO: 35 and 27,
respectively.

[0417] The nucleotide and amino acid sequences of the light chain variable
region of D1 are shown in FIG. 3B and in SEQ ID NO: 39 and 31,
respectively.

[0418] Comparison of the D1 heavy chain immunoglobulin sequence to the
known human germline immunoglobulin heavy chain sequences demonstrated
that the D1 heavy chain utilizes a VH segment from human germline
VH 3-48, a D segment from the human germline 4-23, and a JH segment
from human germline JH 6B. Further analysis of the D1 VH sequence
using the Kabat system of CDR region determination led to the delineation
of the heavy chain CDR1, CDR2 and CD3 regions as shown in FIG. 3A and in
SEQ ID NOs: 3, 7 and 11, respectively.

[0419] Comparison of the D1 light chain immunoglobulin sequence to the
known human germline immunoglobulin light chain sequences demonstrated
that the D1 light chain utilizes a VL segment from human germline
VK L15 and a JK segment from human germline JK 1. Further analysis
of the D1 VL sequence using the Kabat system of CDR region
determination led to the delineation of the light chain CDR1, CDR2 and
CD3 regions as shown in FIG. 3B and in SEQ ID NOs: 15, 19 and 23,
respectively.

[0420] The nucleotide and amino acid sequences of the heavy chain variable
region of E2 are shown in FIG. 4A and in SEQ ID NO: 36 and 28,
respectively.

[0421] The nucleotide and amino acid sequences of the light chain variable
region of E2 are shown in FIG. 4B and in SEQ ID NO: 40 and 32,
respectively.

[0422] Comparison of the E2 heavy chain immunoglobulin sequence to the
known human germline immunoglobulin heavy chain sequences demonstrated
that the E2 heavy chain utilizes a VH segment from human germline
VH 3-48, a D segment from the human germline 4-23, and a JH segment
from human germline JH 6B. Further analysis of the E2 VH sequence
using the Kabat system of CDR region determination led to the delineation
of the heavy chain CDR1, CDR2 and CD3 regions as shown in FIG. 4A and in
SEQ ID NOs: 4, 8 and 12, respectively.

[0423] Comparison of the E2 light chain immunoglobulin sequence to the
known human germline immunoglobulin light chain sequences demonstrated
that the E2 light chain utilizes a VL segment from human germline
VK L15 and a JK segment from human germline JK 1. Further analysis
of the E2 VL sequence using the Kabat system of CDR region
determination led to the delineation of the light chain CDR1, CDR2 and
CD3 regions as shown in FIG. 4B and in SEQ ID NOs: 16, 20 and 24,
respectively.

[0424] Analysis of the framework sequences of the VH and VL
regions of F7, F9, D1 and E2, as compared to the germline sequences from
which they were derived, identified various framework amino acid residues
that differed from germline. Certain framework residues in the N-terminal
regions of the VH and VL segments were chosen for
"back-mutation" to restore the framework residue to the germline
sequence, because these non-germline residues in the N-terminal portion
were encoded by the primers used to create the phage display libraries
described in Example 1. In particular, the following modified forms of
the VH and VL segments of F7, F9, D1 and E2 (referred to as
"GL" forms, for germline) were created using standard molecular biology
techniques to substitute the germline amino acid residue at the indicated
framework position:

[0441] The F7, F9, D1 and E2 Fab fragments are converted to full-length
antibodies using standard recombinant DNA techniques. For example, DNA
encoding the VH and VK regions of one of the Fab fragments can
be cloned into an expression vector that carries the heavy and light
chain constant regions such that the variable regions are operatively
linked to the constant regions. Alternatively, separate vectors can be
used for expression of the full-length heavy chain and the full-length
light chain. Non-limiting examples of expression vectors suitable for use
in creating full-length antibodies include the pIE vectors described in
U.S. Patent Application No. 20050153394 by Black.

Example 3

Binding Characteristics of Anti-CXCR4 Human Monoclonal Antibodies

[0442] In this example, binding characteristics of the anti-CXCR4
antibodies were examined by flow cytometry.

[0443] The human T cell line CEM, which expresses native human CXCR4 on
its cell surface, was used to examine the ability of the F7, F9, D1 and
E2 antibodies to bind to native, cell-surface CXCR4. Full-length F7, F9,
D1 and E2 were titrated in a 1:3 serial dilution series, resulting in a
concentration range from 300 nM to 5 pM. The antibodies were then mixed
with CEM cells and allowed to bind before being detected with a
FITC-conjugated anti-human IgG secondary antibody. The cells were then
analyzed by fluorescent cytometry. The resulting mean fluorescence
intensities are shown in the graph of FIG. 9, which demonstrates that all
four anti-CXCR4 antibodies bind to CEM cells. The EC50 for binding
F7, F9, D1 and E2 were 21 nM, 14 nM, 80 nM and 290 nM, respectively.

[0444] To determine the ability of a panel of anti-CXCR4 antibodies to
compete for binding to CXCR4, competition studies were performed. The
four human anti-CXCR4 antibodies F9, F7, E2 and D1 were used, along with
four commercially available murine monoclonal anti-CXCR4 antibodies
(12G5, 708, 716 and 717; R&D Systems catalog #s: MAB170, MAB171, MAB172
and MAB173, respectively). The anti-CXCR4 antibodies were titrated in a
1:3 serial dilution series resulting in a concentration range from 300 nM
to 5 pM in the presence of a constant concentration of FITC-labeled
anti-CXCR4 antibody F9. The mixture of antibodies was then added to CEM
cells and allowed to bind. The ability of each antibody to compete with
F9 for binding to CEM cells was assessed by fluorescent cytometry and
detection of FITC. The resulting mean fluorescent intensitities are shown
in the graph of FIG. 10, which demonstrates that all seven antibodies
examined (F7, E2, D1, 12G5, 708, 716 and 717) were able to compete with
F9 for binding to CEM cells, although the E2 antibody only demonstrated
partial inhibition at high concentrations compared to the other
antibodies.

[0445] In another set of experiments, the ability of the F7 mAb to bind to
a variety of different cell lines was examined by flow cytometry by
carrying out a FACS titration. Increasing amounts of mAb (from less than
0.001 μg/ml to more than 100 μg/ml) were incubated with 100,00
cells and binding assessed by flow cytometry. The Bmax value also was
determined, which indicates approximately how many CXCR4 molecules are
present on each cell. Based on the binding curves, an EC50 for
antibody binding was determined, the results of which are summarized
below in Table 1:

The results show that F7 mAb is capable of binding effectively to each of
the six cell lines tested, with the lowest EC50s observed with the
Ramos and Raji cell lines. These data also show that the expression of
CXCR4 receptor is highest for Ramos and Namalwa cells and lowest for
MDA-MB-231 cells and DMS79 cells.

[0446] In another binding experiment, the ability of the F7 mAb to bind to
different subsets of human peripheral blood mononuclear cells (PBMCs) was
examined. Human PBMCs were isolated by standard methods and different
cellular subsets were isolated by FACS. In particular, the following
cellular subsets were isolated: (i) CD3.sup.+, (ii) CD20.sup.+; (iii)
CD11b.sup.+ and (iv) CD14.sup.+. Flow cytometry experiments conducted
with the F7 mAb (at 33 μg/ml) demonstrated that the F7 mAb was capable
of binding effectively to each of the four subsets, as compared to an
isotype-matched control antibody.

Example 4

Inhibition of SDF-1 Binding to CXCR4 by Anti-CXCR4Antibodies

[0447] To determine the ability of the anti-CXCR4 human antibodies to
inhibit the binding of SDF-1 to CXCR4, a competition study was performed
using 125I-labeled SDF-1 and CEM cells, which naturally express
CXCR4. A comparison of anti-CXCR4 antibodies on blocking SDF-1 binding to
CEM cells was performed by a standard radio-labeled ligand binding assay.
The anti-CXCR4 antibodies were serially diluted 1:3 to yield a range of
concentrations from 300 nM to 137 pM. The antibodies were added to
750,000 CEM cells in 100 μl in the presence of 100 pM 125I-SDF-1
with a specific activity of 2000 Ci/mmole (Amersham, catalog
#IM314-25UCI). An irrelevant antibody of the same isotype was used as a
negative control. The total possible bound radio-labeled ligand was
determined by allowing the 125I-SDF-1 to bind to CEM cells in the
absence of antibodies for 2 hours at 4° C. Non-specific binding of
the radio-labeled ligand was determined by allowing the 125I-SDF-1
to bind in the presence of 1 μM unlabeled SDF-1 (Peprotech, catalog
#300-28A). The amount of cell-associated 125I-SDF-1 was determined
by standard methods. The results are shown in FIG. 11, which demonstrates
that the F7 antibody provides the most effective blockade of SDF-1
binding to CXCR4 expressed on CEM cells. The F9 and D1 antibodies also
blocked SDF-1 binding, although more moderately than F7. The E2 antibody,
although it does bind to CXCR4 on CEM cells (as demonstrated in Example
3), did not effectively block SDF-1 binding to CXCR4 on CEM cells. The
EC50s for SDF-1 blockade by F7, F9 and D1 were 2.3 nM, 12.5 nM and
28.6 nM, respectively.

Example 5

Inhibition of SDF-1-Induced Calcium Flux by Anti-CXCR4 Antibodies

[0448] To determine the ability of the anti-CXCR4 human antibodies to
inhibit calcium flux in CEM cells induced by SDF-1, CEM cells were first
labeled with the fluorescent dye Calcium 3 (Molecular Devices). The
anti-CXCR4 antibodies were titrated in a 1:3 serial dilution series
resulting in a concentration range from 100 nM to 1 pM and allowed to
bind to 200,000 CEM cells in 200 μl and incubated 10 minutes at room
temperature prior to loading into a Flexstation machine (Molecular
Devices). As a negative control, an irrelevant antibody of the same
isotype was used. Cells were then stimulated with a final concentration
of 50 nM recombinant human SDF-1α (Peprotech), added as 500 nM in a
volume of 22 μl for a final volume of 222 μl. The resulting calcium
flux was measured for 200 seconds per well. As a positive control, cells
in the absence of antibody were stimulated with SDF-1α (made up in
Hank's buffered saline (HBS) with 0.1% BSA or HBS) to achieve a maximum
possible calcium flux signal. To determine a baseline, cells were
stimulated with HBS with 0.1% BSA. The SDF-1α-stimulated release of
calcium was measured by the development of calcium-dependent
fluourescence over time. The area under the curve of the resulting
fluorescence trace was reported as an indication of calcium flux. The
resulting inhibition of calcium flux by the anti-CXCR4 antibodies is
represented in FIG. 12. The data were plotted and the EC50s were
calculated using GraphPad Prism software and the non-linear curve fit,
sigmoidal dose response formula. Antibodies F7, F9 and D1 inhibited
SDF-1α-induced calcium flux. Antibody E2, although it did bind to
CXCR4 (as demonstrated in Example 3), did not significantly inhibit
SDF-1α-induced calcium flux. The EC50s for inhibition of
SDF-1-induced calcium flux by F7, F9 and D1 were 0.90 nM, 0.32 nM and
0.57 nM, respectively.

Example 6

Inhibition of SDF-1-Induced Migration of CEM Cells by Anti-CXCR4Antibodies

[0449] To determine the ability of the anti-CXCR4 human antibodies to
inhibit migration of CEM cells induced by SDF-1, CEM cells first were
labeled with the BATDA reagent (Perkin Elmer). The anti-CXCR4 antibodies
were titrated in a 1:3 serial dilution series resulting in a
concentration range from 100 nM to 1 pM and allowed to bind to labeled
CEM cells at a density of 10 million cells per ml. As a negative control,
an irrelevant antibody of the same isotype was used. Recombinant human
SDF-1α (Peprotech) was added at 5 nM at 30 μl per well to the
lower chamber of a 96 well Neuroprobe migration plate with 5.7 mm
diameter filters per well. Each well contains 5 μM pores. Labeled CEM
cells with and without antibody were loaded onto the filters at a
concentration of 0.5 million cells per well in a volume of 50 μl. The
migration plate was incubated at 37° C. for 2.5 hours. Migrated
cells were captured in the lower chamber of the plate, lysed and detected
with Europium detection solution (Perkin Elmer). The chemi-luminescent
signal was recorded on a Fusion instrument. The resulting inhibition of
SDF-1α-induced migration by the anti-CXCR4 antibodies in shown in
FIG. 13. The results demonstrated that antibodies F7 and F9 inhibited
migration effectively, while antibodies D1 and E2 did not significantly
inhibit migration. The EC50s for inhibition of SDF-1-induced CEM
cell migration by F7 and F9 were 12.44 nM and 18.99 nM, respectively.

Example 7

Inhibition of HuVEC Capillary Tube Formation by Anti-CXCR4 Antibodies

[0450] In this example, the ability of the anti-CXCR4 human antibodies to
inhibit capillary tube formation by human umbilical vein endothelial
cells (HuVEC) was examined. Matrigel was diluted 1:1 with RPMI and plated
onto the wells of a 96 well plate and allowed to polymerize for 30
minutes at 37° C. HuVEC (from Cambrex, cat. # CC-2519) at 80%
confluence were trypsanized and resuspended at 1×106 cells per
ml in RPMI with 0.5% FBS. Antibodies were well mixed with HuVEC at a
final concentration of 3 μg/ml and allowed to incubate at room
temperature for 30 minutes. An irrelevant antibody of the same isotype or
media alone was used as a negative control. As a positive control of
inhibition of tube formation, a mouse anti-human αvβ3
(CD51/CD61) antibody (R&D Systems, cat. # MAB3050) was used. HuVEC with
or without antibodies were plated onto the matrigel-coated wells and
incubated at 37° C. for 18 hours.

[0451] The HuVEC incubated with media alone or with the isotype-matched
control antibody formed capillary tubes resulting in the appearance of
connected cells across the plate with 3-5 points of connection or branch
points per cell. The HuVEC incubated with either the anti-CXCR4 human
antibodies or the anti-αvβ3 antibody did not form capillary
tubes. The cells appeared isolated and with few or no branch points. The
anti-CXCR4 antibodies that were most effective in blocking SDF-1 binding,
SDF-1-induced calcium flux and SDF-1-induced migration, namely F7 and F9,
were also the most effective in inhibiting capillary tube formation. The
anti-CXCR4 antibody E2, whch binds to CXCR4 but does not block SDF-1
binding or SDF-1-induced effects, did not inhibit capillary tube
formation.

Example 8

Inhibition of Tumor Cell Proliferation In Vitro by Anti-CXCR4 Antibodies

[0452] In this example, the ability of the anti-CXCR4 human antibodies to
inhibit proliferation of Ramos tumor cells (a human Burkitt's lymphoma
cell line) in vitro was examined. In the assay, 1×104
cells/well were incubated with increasing doses (10-3 to 300 nM) of
F7 IgG4 antibody, F9 IgG1 antibody, E2 IgG1 antibody, F9 Fab' antibody or
isotype controls. The cells were incubated with antibody for 72 hours,
with 3H-thymidine being added for the final 24 hours of incubation
to allow for monitoring of cell proliferation. Following the incubation,
incorporation of 3H-thymidine by the cells was measured by standard
techniques. The results are shown in the graph of FIG. 14. The results
demonstrate that the F7 IgG4, F9 IgG1 and E2 IgG1 antibodies each were
able to inhibit Ramos cell proliferation, as indicated by decreased
3H-thymidine incorporation when incubated with these antibodies,
whereas the F9 Fab' fragment did not inhibit cell proliferation. These
results indicate that the anti-CXCR4 human antibodies have a direct
anti-proliferative effect on the tumor cells in vitro and thus do not
require secondary cross-linking to achieve an anti-proliferative effect.

[0453] In this example, the ability of the anti-CXCR4 human antibodies to
inhibit proliferation of an established solid tumor in vivo was examined
using a Ramos subcutaneous tumor cell model. In this assay,
10×106 Ramos cells/mouse were implanted into the flank region
of each mouse and allowed to grow to a mean size of 40 mm3,
calculated by length×width×height/2 of the tumors. The mice
then received an intraperitoneal (i.p.) injection of a first dose of
antibody (designated as day 0 of treatment) and received a second i.p.
dose of antibody on day 7. Mice treated with a Fab' fragment antibody
also received i.p. antibody doses on day 3 and day 10. Groups of mice
(n=8) were treated with either (i) vehicle; (ii) isotype control (15
mg/kg); (iii) F7 IgG4 (15 mg/kg); (iv) F9 IgG1 (15 mg/kg); (v) F9 Fab'
(10 mg/kg); or (vi) anti-CD20 positive control (15 mg/kg). Tumor volume
and mouse body weight were measured at regular intervals (approximately
2-3 times/week) between day 0 and day 30 post dosing. The results of the
experiment are presented in FIGS. 15A, 15B and 15C, which show mean tumor
volume (FIG. 15A), median tumor volume (FIG. 15B) and median % body
weight change (FIG. 15C). The results demonstrated that, like the
positive control, the F7 IgG4 and F9 IgG1 antibodies significantly
inhibited tumor cell growth as measured by increased tumor volume,
whereas the F9 Fab' fragment did not inhibit tumor cell growth as
compared to the isotype control. All treatments were well-tolerated as
indicated by no significant body weight change. The differences in body
weights between treatments was most likely due to the weights of the
tumors. The results indicate that the anti-CXCR4 human antibodies are
capable of inhibiting growth of an established solid tumor in vivo.

Example 10

Increased Survival Time in a Mouse Systemic Tumor Cell Model by Treatment
with an Anti-CXCR4Antibody

[0454] In this example, the ability of an anti-CXCR4 human antibody to
increase survival time of mice was examined using a Ramos systemic tumor
cell model. In this assay, 1×106 Ramos cells/mouse were
injected intravenously (i.v.) into each mouse on day 0. The mice then
received an intraperitoneal (i.p.) injection of a first dose of antibody
on day 1 (i.e., one day after i.v. administration of tumor cells) and
received four more i.p. doses of antibody, on days 5, 8, 15 and 22 (mice
treated with the positive control antibody were treated only on day 1).
Groups of mice (n=8) were treated with either (i) vehicle; (ii) isotype
control (15 mg/kg); (iii) F9 IgG1 (15 mg/kg); or (iv) anti-CD19 positive
control (15 mg/kg). Percent survival was measured at regular intervals
between day 0 and day 50 post dosing (hind leg paralysis was used as the
endpoint of the experiment). The results of the experiment are presented
in FIG. 16, which shows percent survival over time. The median # days of
survival for the mice treated with either vehicle or the isotype control
were 23 and 25.5 days, respectively, whereas the median # days of
survival of the mice treated with one dose of the anti-CD19 positive
control was 39 days. Significantly, 100% of the mice in the group treated
with five doses of the F9 IgG1 antibody survived to the end of the
experiment. These results indicate that the anti-CXCR4 human antibody is
capable of increasing survival times of mice in a systemic tumor cell
model.

Example 11

Induction of Apoptosis by Anti-CXCR4 Monoclonal Antibody F7

[0455] In this example, the ability of the anti-CXCR4 mAb F7 to induce
apoptosis in different cells was examined. In the apoptosis assay, F7 mAb
at 10 μg/ml was incubated with either Ramos cells (500,000 cells),
Namalwa cells (500,000 cells) or R1610 cells transfected to express CXCR4
(100,000 cells) Untransfected R1610 cells were used as a negative
control. Anti-CXCR4 mAb F7 or isotype control antibody was incubated with
cells at 37° C. and 250 μl samples were removed at 24, 48 and
72 hours. To assess apoptosis, the cells from various time points were
incubated with Annexin V-FITC-FL1 and Propidium Iodide--FL3, followed by
flow cytometry. The combined percentage of cells collected in the FL1,
FL3 and FL1-FL3 double positive quadrants were considered apoptotic. To
remove background, the percentages of isotype antibody-induced apoptotic
cells was subtracted from the percentage of F7 mAb-induced apoptotic
cells.

The results demonstrate that the F7 mAb is capable of inducing apoptosis
in the Ramos, Namalwa and R1610-CXCR4 cells while F7 had no effect on
induction of apoptosis of parental R1610 cells indicating that the
response was CXCR4-specific.

[0458] In this example, the ability of anti-CXCR4 human antibodies to
inhibit proliferation or induce apoptosis of established solid tumors in
vivo was examined using additional tumor cell models similar to the Ramos
model described above in Example 9. A variety of tumor cell lines were
examined. Representative experiments and results are as follows.

[0459] In one experiment, 7.5×106 MDA-MB231 human breast cancer
cells/mouse were implanted into the flank region of each mouse and
allowed to grow to a mean size of 100 mm3, calculated by
length×width×height/2 of the tumors, which was day 7
post-implantation. The mice were randomized into different treatment
groups and received an intraperitoneal (i.p.) injection of a first dose
of antibody on day 7 post-implantation, received a second i.p. dose of
antibody on day 14 post-implantion and then received a third dose on day
46 post-implantation. Groups of mice (n=9) were treated with either (i)
vehicle (PBS); (ii) IgG1 isotype control (15 mg/kg); (iii) IgG4 isotype
control (15 mg/kg); (iv) F7 IgG1 (15 mg/kg); or (v) F7 IgG4 (15 mg/kg).
Tumor volumes were measured at regular intervals and the mean and median
tumor volume determined for each treatment group at each interval. The
results of this experiment are summarized below in Table 3, which shows
mean tumor volume (in mm3) and % tumor growth inhibition (TGI) at
day 52, and median tumor volume (in mm3) and % TGI at day 59
post-implantation:

Additionally, one of the mice in the F7 IgG4 treatment group was tumor
free at day 59. The results demonstrate that the F7 mAb is capable of
inhibiting growth of MDA-MB231 breast cancer cells in vivo.

[0460] In a second experiment, 5×106 DMS79 human small cell
lung carcinoma cells/mouse were implanted into the flank region of each
mouse and allowed to grow to a mean size of 160 mm3, calculated by
length×width×height/2 of the tumors, which was day 7
post-implantation. The mice were randomized into different treatment
groups and received intraperitoneal (i.p.) injections of antibody on a
dosing schedule of Q3D×5 (every three days for five times). Groups
of mice (n=10) were treated with either (i) vehicle (PBS); (ii) IgG4
isotype control (10 mg/kg); or (iii) F7 IgG4 (10 mg/kg). Tumor volumes
were measured at regular intervals and the mean and median tumor volume
determined for each treatment group at each interval. The results of this
experiment are summarized below in Table 4, which shows mean and median
tumor volume (in mm3) and % tumor growth inhibition (TGI) at day 34
post-implantation:

The results demonstrate that the F7 mAb is capable of inhibiting growth
of DMS79 human small cell lung carcinoma cells in vivo.

[0461] Additional subcutaneous xenograft tumor models were tested for the
ability of anti-CXCR4 antibodies to inhibit tumor growth, in experiments
similar to those described above and in Example 9. In an experiment using
SU-DHL-6 B cell lymphoma cells, the results showed that treatment with
the F7 IgG4 mAb at 15 mg/kg resulted in approximately 60% tumor growth
inhibition. Similarly, in an experiment using Namalwa Burkitt's lymphoma
cells, the results showed that treatment with the F7 IgG4 mAb at 3 mg/kg
resulted in approximately 70% tumor growth inhibition. In contrast, no
tumor growth inhibition by the F7 mAb was observed in experiments using
NIH-H226 lung carcinoma cells or HPAC human pancreatic adenocarcinoma
cells. However, staining of these cells by the F7 mAb in flow cytometry
experiments showed minimal in vitro expression. Although the tumor cells
in vivo were stainable by the mAb by immunohistochemistry, it is unclear
at what stage of their tumor growth CXCR4 began to be expressed. This
suggests that experession of CXCR4 by these two cell lines was
insufficient to allow for tumor growth inhibition or induction of
apoptosis in vivo by anti-CXCR4 treament.

Example 13

Inhibition of Lung Metastases In Vivo by Anti-CXCR4Antibodies

[0462] In this example, the ability of the F7 anti-CXCR4 mAb to inhibit
lung metastases was examined using a C57 mouse systemic tumor model. More
specifically, 0.4×106 B16-CXCR4 cells (B16 cells transfected
to express human CXCR4) were injected intravenously into each of 30 mice
of the C5-7 strain. The mice were randomized into three groups of
ten mice each, which were then treated with either (i) vehicle (PBS);
(ii) IgG4 isotype control (5 mg/kg); or (iii) F7 IgG4 (5 mg/kg). The
antibody or vehicle was injected intraperitoneally 30 minutes after the
B16-CXCR4 cells were injected intravenously. Lungs were harvested on day
14 and the number of lung metastatis nodules was quantitated. The results
are summarized below in Table 5, which shows the mean and median number
of lung metastases in each group:

The results show that treatment with the F7 mAb led to a reduction in the
mean number of lung metastatic nodules of 56%, whereas reduction was only
15% with the isotype control antibody, demonstrating that the F7 mAb is
capable of inhibiting lung metastases in a systemic tumor model.